BIOLOGIA MOLECOLARE - CPM SAS · quality control in molecular biology The use of the Polymerase Chain Reaction (PCR) and the NAT tech-nologies in molecular diagnostic has increased

BIOLOGIA MOLECOLARE

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Quality control in molecular biology

Complete kitsHuman diagnosticsVirusBacteriaProtozoaGeneticOncologyFood diagnosticsVeterinary diagnostics

Single componentsExtraction kitMaster mixPositive control

Molecular diagnosisAutomated DNA sequencerReal time PCR

CLONpact and CLONgel

Ordering information

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molecular biology in diagnosticsRecent developments in molecular biology have provided clinical-laboratories with new methods to place alongside traditionalresearch techniques, so contributing to the improvement of thediagnosis and prognosis of major pathologies.In some cases tech-niques of molecular biology are the only avail-able means ofmaking diagnoses in hereditary diseases (for exam-ple, the identifi-cation of healthy carriers of genetic mutations).In forensic medici-ne (paternity or biological identity analysis) and in virology (the dis-crimination of viral strains or variants not identifiable by othermethods).One of the techniques that has made this innovationpossible in clin-ical laboratory diagnostics is the Polymerase ChainReaction (PCR). It enables analysts to obtain billions of copies of agiven DNA region, which in turn makes it possible to detect anypathogenic micro-or-ganism present in a biological sample, even inthe absence of infec-tion indicators such as antibodies, or to carryout a simple molecular analysis of a given genome section as requi-red in genetic and fo-rensic medicine investigations.The PCR is so sensitive that it is theore-tically possible to detect a single targetDNA sequence.This with the highdegree of specificity of the technique,means that PCR can be applied in allfields of biology, particularly laboratorydi-agnostics, where commercial kitshave simplified operating proce-duresand, where possible, their automation.This brochure has been drawn up topresent the infective agents that canbe detected in biological samples bygene amplification kits for the identifi-cation of specific DNA and RNA sequences. These prod-ucts allow theidentification of the genomes of themost important viruses and pathoge-nic micro-organisms, leading to thedirect and highly sensitive diagnosis ofinfections under way.Most of the Clonit’s kits use NESTEDPCR, this procedure was cho-senbecause of its superior sensitivity andspecificity over those from tests with asingle target sequence.Clonit’s products do not present any infection risks for the users.

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clonit’s productsClonit’s products are molecular biology systems ranging from the screening and the typing of viruses, bacteria and protozoa. The products range is very wide and can satisfy all the requests of the diagnostic laboratory.The panel products is very flexible and involves complete kit as well as single components.In the complete kit all the steps of the procedure are avilable with ready to use solutions:

Ω nucleic acid extraction

Ω ready-to-use solutions for single or nested amplification

Ω positive and negative control

Ω enzymes

Ω sample gel-loading solution

Ω electrophoresis buffer

Ω pre-cast pre-stained gels

The shipment of enzymes is in a separate package at –20°C with dry ice, where dry ice is accepted.Please note that all the products in this catalog, already CE marked according to european regulation 98/79/CE, are identified with the

symbol.

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quality control in molecular biology

�quality control in molecular biology

real time standardsHepatitis B virus (HBV-DNA complete genome)Hepatitis C virus (HCV-DNA 5’UTR)Human Immunodeficiency Virus (HIV-DNA gag)Citomegalovirus (CMV-IEA)Epstein-Barr Virus (EBV – Bam HI-W)Human Papilloma Virus (HPV16)Human Papilloma Virus (HPV18)Mycobacterium tuberculosis IS6110Hepatitis C virus (HCV-RNA 5’UTR)Severe acute respiratory syndrome (SARS-RNA)

run controlHepatitis B virus (HBV-DNA Complete genome)Hepatitis C virus (HCV-RNA 5’UTR)Human Immunodeficiency Virus (HIV-RNA gag)

quanti controlHepatitis B virus (HBV-DNA Complete genome)Hepatitis C virus (HCV-RNA 5’UTR)Human Immunodeficiency Virus (HIV-RNA gag)Citomegalovirus (CMV-IEA)

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quality control in molecular biologyThe use of the Polymerase Chain Reaction (PCR) and the NAT tech-nologies in molecular diagnostic has increased to the point where in is now accepted as the gold standard for detecting nucleic acid s from a number of origins and it has become an essential in the research laboratory.In the Molecular Biology field one of the most important item in-volves the use of standards and calibrators.Clonit prepared a wide series of synthetic standards (RNA and DNA) for the control and calibration of all the molecular biology appli-cations. The plasmidic nature of Clonit’ controls bring a series of advantage and have no problems of infectivity. The controls are cali-brated, when available, to the WHO international standards.Clonit’ quality controls aim to achieve three main goals.

1 “Real time standards”� “Run Control” � “Quanticontrol”

The run control is preparated with an RNAase free serum that allow the operator to handle the controls as an unknows sample, and ceck all the steps of the test from the extraction to detection.

quality controlin molecularbiology


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Code Product description Test

05960209 HCV 5’ UTR - DNA(REAL TIME)

8x35µl8 vials 1x106 cps/µl (red) + 8 vials 1x105 cps/µl (blu) + 8 vials 1x104 cps/µl (green) + 8 vials 1x103 cps/µl (yellow)

05960329 HIV gag(REAL TIME)


05960116 HBV complete genome(REAL TIME)


05960467 CMV IEA(REAL TIME)


05960468 EBV Bam HI-W(REAL TIME)


05960469 HPV 16 genome(REAL TIME)


05960470 HPV 18 genome(REAL TIME)


05960564 MTB IS6110(REAL TIME)


05960210 HCV 5’ UTR - RNA(REAL TIME)


05960471 SARS - RNA(REAL TIME)


real time standards“Real time standards” are for the reference curves in the quantitative analysis with real time PCR.


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run control“Run Control” is for the validation of each amplification run with the Polymerase Chain Reaction (PCR).


05960124 HBV complete genome(quanticontrol)

8x15µl8 vials 1x104 IU/ml (red) + 8 vials 1x103 IU/ml (green) + 8 vials 1x102 IU/ml (yellow)

05960217 HCV 5’ UTR - RNA(quanticontrol)

8x15µl8 vials 2x104 IU/ml (red) + 8 vials 2x103 IU/ml (green) + 8 vials 6x102 IU/ml (yellow))

05960333 HIV gag - RNA(quanticontrol)

8x15µl8 vials 1000 cps/µl (red) + 8 vials 100 cps/µl (green) + 8 vials 10 cps/µl (yellow)

05960488 CMV IEA(quanticontrol)

8x15µl8 vials 100 cps/µl (red) + 8 vials 20 cps/µl (green) + 8 vials 5 cps/µl (yellow)


05960117 HBV complete genome(run control)

24x15µl24 vials 5x103 cps/µl

05960215 HCV 5’ UTR - RNA(run control)

24x15µl24 vials 1000 cps/µl

05960332 HIV gag - RNA(run control)


05960487 CMV IEA(run control)


quanti control“Quanticontrol” is for the assessment of sensivity and reproducibility of all commercial and “home brew” systems


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complete kits

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human diagnostics

1�complete kits / human diagnostics

VirusHepatitis B virus (HBV)Hepatitis C virus (HCV)Human Immunodeficiency Virus (HIV)Citomegalovirus (CMV)Epstein-Barr Virus (EBV)Human Papilloma Virus (HPV)Parvovirus B19 (B19V)Herpes Simplex Virus (HSV 1)JC Virus (JCV)Poliovirus (PV)Varicella Zoster Virus (VZV)Severe acute respiratory syndrome (SARS)

BacteriaMycobacterium tuberculosisHelicobacter pyloriChlamydia trachomatisChlamydia pneumoniaeMycoplasma pneumoniaeLegionella pneumophilaBorrelia burgdorferiPeriodontal Disease MultiplexListeria monocytogenesE.coli 0157:H7SalmonellaBacillus anthracis

ProtozoaPlasmodiumLeishmaniaToxoplasma gondii

GeneticFactor V LeidenFactor II G20210AMTHFR C677T

OncologyMelanoma

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25

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hepatitis H virus (HBV)This test shows up the presence of the hepatitis B virus (HBV).The virus is a cause of post-transfusion hepatities and one of the main causes of chronic hepatitis that, through the activation of phe-nomena in the immune system and immunocomplex lesions, may trigger a chronic hepatic condition resulting in cirrhosis and hepa-tomas. The incidence of hepatitis B is rising up in intravenous drug abusers, between whom infection is transmitted parenterally.The infection may also be passed from mother to newborn child both through the placenta and at birth.People most exposed to risk are therefore patients undergoing multiple transfusions and those professionally exposed, such as doctors, nurses and dentists.The disease has an incubation period of up to six months. Many sub-jects may remain perfectly healthy even though they carry the virus and represent the most important source of infection.Early diagnosis of infection is therefore of the highest importance, and is made extremely easy by PCR.The HBsAg kit uses specific primers for the superficial antigen, while the HBcAg uses primers for core antigen.


AMS01 HBV- HBsAg gene qualitative kit 24 test

AMS01/F HBV- HBsAg gene qualitative kit 96 test

AMS01Q HBV- HBsAg gene semi-quantitative kit 24 test

AMS02 HBV- HBcAg gene qualitative kit 24 test

AMS16 HBV-HBcAg pre-core mutation qualitative kit 24 test

AMS92 HBV-HBsAg (“nested”) qualitative kit 24 test

AMS92/F HBV-HBsAg (“nested”) qualitative kit 96 test

complete kits / human diagnostics / virus

virus

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hepatitis C virus (HCV)This test shows up the presence of the hepatitis C virus (HCV), which is responsible for 65-90% of new hepatitis diagnoses. The infection is transmitted parenterally and the disease is generally mild in form or asymptomatic in its acute phase (50-70%) of cases, with a high percentage of development into chronic forms.The clinical course is usually erratic, with fluctuating transaminase values which can lead to innaccurate assessments of the develop-ment of the disease and wrong diagnoses for treatment.The diagnostic instrument provided by PCR has the invaluable ad-vantage of diagnosing the disease at an early stage and distinguish-ing it from other forms of hepatitis, which serological parameters alone are unable to do with any great reliability.A nested primer pair is used to specifically amplify a sequence of the 5’ UTR region.


AMS14 HCV-RNA 5’UTR (RT+”nested”) qualitative kit 24 test

AMS14/F HCV-RNA 5’UTR (RT+”nested”) qualitative kit 96 test

AMS14Q HCV-RNA 5’UTR (RT+“nested“) Semi-quantitative kit 24 test


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citomegalovirus (CMV)This test shows up the presence of the CMV, which belongs to the Herpesviridae family. CMV is pathogenically limited, but may cause serious systemic infections in those who have inhibited immune de-fence systems.CMV pathology is currently considered highly important since it frequently strikes AIDS patients and subjects who have undergone organ transplants, in which it is responsible for clinical conditions marked by a high mortality rate. In pregnant women there is also a danger of transmission from mother to foetus, resulting in miscar-riage or foetus malformations such as mental or motor retardation, atrophy of the optic nerve, paraventricular calcification and karyor-etinitis.The presence of antibodies in mothers, even before pregnancy, does not ensure an adequate immune defence.According to recent estimates, one child on three thousand suffers from pathologies caused by CMV.The disease is transmitted parenterally or by direct contact with in-fected human secretions. It is generally asymptomatic and develops as a latent infection with a persistence of the viral genome in a num-ber of cells, which may lead to viral reactivation, even when the virus has been eliminated for years.A nested primer pair is used to specifically amplify a sequence of the “immediate early region“.


AMS12 CMV (“nested”) qualitative kit 24 test

AMS12/F CMV (“nested”) qualitative kit 96 test

AMS12Q CMV (“nested”) Semi-quantitative kit 24 test


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AMS03 HIV-DNA gag gene (“nested”) qualitative kit 24 test

AMS03/F HIV-DNA gag gene (“nested”) qualitative kit 96 test

AMS04 HIV-DNA env gene (“nested”) qualitative kit 24 test

AMS04/F HIV-DNA env gene (“nested”) qualitative kit 96 test

AMS47 HIV-RNA gag gene (RT + “nested”) qualitative kit 24 test

AMS47/F HIV-RNA gag gene (RT + “nested”) qualitative kit 96 test

AMS47Q HIV-RNA gag gene (RT + “nested”) semi-quantitative kit 24 test

human immunodeficiency virus (HIV)This test shows up the presence of the AIDS virus (HIV). HIV infection is marked by a variable latent period, which is sometimes very long, between contact with the virus and the appearance of the first clini-cal symptoms of the illness. During this period an infected individual may unwittingly infect other people.It is therefore of primary importance to be able to diagnose HIV in-fection before symptoms are manifested. As antibody response to HIV proteins develops over a period of several months after infection, normal methods of anti-HIV antibody detection carried out in this period are not able to show up infection. The PCR method makes it possible to identify, with a high degree of sensitivity, specific HIV proviral DNA sequences before the relevant antibodies appear.The clinical applications of this technique include:

Ω Diagnosis in newborn babies born to HIV positive mothers.

Ω Diagnosis in subjects with doubtful results in the specific antibody dosage.

Ω Diagnosis in high-risk HIV-negative subjects who may be in the window period.

Ω Identification of HIV in specific tissue (bioptic samples, tissue in paraffin, etc..).

Ω The kits use nested sets of primers that amplify a sequence of the “gag” gene and “env” gene.


1�complete kits / human diagnostics / virus


AMS13 EBV (“nested”) qualitative kit 24 test

AMS13/F EBV (“nested”) qualitative kit 96 test

AMS13Q EBV (“nested”) Semi-quantitative kit 24 test

epstein-barr virus (EBV)This test shows up the presence of the EBV, the principal etiological agent of infectious mononucleosis (IM) as well as a contributory fac-tor in the etiology of Burkitt’s lymphoma and nasopharyngeal car-cinoma. It has a worldwide distribution, such that 80 to 90 % of all adults have been infected.Primary infections occur during the first decade of life in areas with crowded living conditions and poor hygiene.Childhood infections are mostly asymptomatic, but infrequently may be associated with classical IM. In contrast, 50 to 75% of young adults undergo pre-symptomatic primary EBV infections, with illness ranging from mild to severe.As with other Herpes viruses, EBV causes a persistent latent infec-tion which can be reactivated under immunosuppression. Reactived infections are generally asymptomatic.Clonit’s nested PCR kit is based on a nested amplification of the Bam-HIW region sequence.


human papilloma virus (HPV)This test shows up the presence of the HPV total and serotypes 6, 11, 16, 18, and 33.HPV is know to be a sexually-transmitted etiological agent causing genital condylomas, laryngeal papillomas, intraepithelial cervical neoplasias and carcinomas of the human ano-genital tract. About 60 types of HPV are known to exist.HPV 6 and 11 are easily recognizable such as condylomas and papil-lomas and, rarely, in intraepithaelial cervical neoplasias. HPV 16 and 18 have been observed mainly in highly cervical carcinomas.Besides identifying the virus, therefore, it is extremely important to ascertain the HPV serotype in order to obtain an accurate diagnosis of the lesion and a timely intervention with the appropriate therapy.


AMS07 HPV total qualitative kit 24 test

AMS08 HPV 16 qualitative kit 24 test






parvovirus B1� (B1�V)This test shows up the presence of Parvovirus B19, an icosahedral, non-enveloped DNA virus with a diameter of between 18 and 26 nm.Parvovirus B19 was discovered in England in 1975 in a panel of blood samples. Sample number “B19” contained a Parvovirus-like particle which was later found to be the causative agent of Erythema Infec-tiosum. This human Parvovirus was originally named Parvovirus B19. In 1995, Human Parvovirus B19 was further classified as an Erythro-virus, and renamed “B19 virus” or “B19V”. Since it’s discovery it has been associated with a wide and ever expanding range of clinical syndromes.Parvovirus B19 causes several syndromes, including Erythema In-fectiosum, chronic arthritis in adults, aplastic crisis in patients with haemolytic anemias, foetal death, and chronic anaemia and neutro-penia in immunocompromised patients.Parvovirus B19 infection is transmitted primarily through respiratory secretions. It can also be transmitted through infected serum via blood transfusions, transplantation or transplacentally from mother to foetus.The virus targets human erythroid progenitor cells in the bone mar-row and spleen. These cells then lyse causing a decline in the eryth-rocyte count and haemoglobin concentration resulting in severe anaemia. There are also declines in the lymphocyte, granulocyte and platelet counts.PCR kit is based on the amplification of the region encoding the structural proteins VP1 (84 kDa).


AMS17 Parvovirus B19 qualitative kit 24 test

�0complete kits / human diagnostics / virus

herpes simplex virus (HSV 1-�)This test shows up the presence of HSV, infections in newborn ba-bies and other clinical manifestations.HSV genital infection, which may be involved in up to 40% of primary genital lesions, may be asymptomatic or present an unclear clinical profile, and be transmitted to new-born babies.The latter may present a number of clinical profiles, generally sys-temic in character, affecting the central nervous system. This carrier a high mortality rate and high incidence of neurological and ocular complications.Less serious infections caused by HSV include aphthous stomatitis, Vincent’s stomatitis, herpetic eczema, kerato-conjunctivitis and her-pes labialis. Infections may be primary in patients with no antibod-ies, or recurrent.In the latter case relapses are caused by specific stimuli such as excessive stress, menstruation or excessive exposure to the sun.The kit uses a set of primers that amplify a sequence of the gene coding for gD protein.


AMS18 HSV 1-2 (“nested”) qualitative kit 24 test

AMS18/F HSV 1-2 (“nested”) qualitative kit 96 test

�1complete kits / human diagnostics / virus

JC virus (JCV)This test shows up the presence of the JC virus, a member of the family Papovaviridae.Named after the initials of the patient from which virus was isolated in 1970, JC virus causes persistent infection in the urinary tract and Progressive Multifocal Leukoencephalopathy (PML), a severe demy-elinating disease of the central nervous system. It is characterised by dementia, muscular weakness affecting one side of the body, speech and vision disruption and progression to death.PML has become more frequent since the onset of Aids epidemic. JC virus, responsible for this disease, has been much studied dur-ing the last years. Numerous physiopathologic aspects of JC virus infection have been elucidated, but this virus is far from having revealed all its secrets. The transmission route remains hypothetical although the respira-tory route seems the most likely and signs of primary infection are totally unknown. It is well established that both B lymphocytes and the other peripheral blood cells play a major role in body dissemina-tion at a very early stage of the disease.This virus produces latent and chronic infections and the viral DNA is integrated into cellular chromosomes of transformed cells.The nested amplification is based on the “early” region sequence coding large t-antigen andsmall t-antigen.


AMS24 JCV (“nested”) qualitative kit 24 test

��complete kits / human diagnostics / virus

poliovirus (PV)This test shows up the presence of Poliovirus (PV) and identifies the three different PV genotypes: PV-1, PV-2, PV-3.Poliovirus, a RNA genome virus, belongs to the genus Enterovirus (Picornaviridae) and it is the agent that causes poliomyelitis.The effective control of poliomyelitis was achieved by the introduc-tion, about 30 years ago, of two polio vaccines, inactivated (IPV) and oral(OPV). The oral vaccine contains live, attenuated poliovirus strains. The massive administration of the OPV in large areas of the world considerably altered the distribution of PV variants in nature.Contrary to what might have been expected, once diffused in the human population, the vaccine strains not only failed to completely replace wild viruses, but they themselves underwent variation dur-ing intra- and inter-human passages.Despite the morbidity of poliomyelitis, several problems still trouble our understanding of the epidemiology of the disease.The characterization of PV strains with Clonit’s RT-PCR kits is based on the amplification of the region coding VP1 capsid polypeptide, and the restriction analysis of the antigenic site 1.


AMS46 Poliovirus(RT + single amplification)

qualitative kit 24 test


varicella zoster virus (VZV)This test shows up the presence of the virus Varicella zoster, respon-sible for Varicella, or chicken-pox.This pathology is particularly dangerous for a foetus if the mother contracts the disease during the first three months of pregnancy since it causes congenital malformations.New-born babies can also contract the infection and suffer enceph-alitis or serious damage to the nervous system.The disease produces a permanent immune response which protects individuals from further infection. The virus enters the body mainly through the mucus in the respiratory system and is released into the bloodstream. It is also found in the coetaneous lesions it causes.A single primer pair is used to specifically amplify a sequence of the C region within the VZV genome.


AMS23 VZV qualitative kit 24 test


severe acute respiratory syndrome (SARS)This test show up the presence of the SARS coronavirus genome, which is responsible of severe acute respiratory syndrome. Severe acute respiratory syndrome is a infective patology identified in 2003, reported in Hanoi, Hong Kong, Singapore. In general, SARS begins with a high fever (temperature greater than 38.0°C). Other symptoms may include headache, an overall feeling of discomfort, and body aches. Some people also have mild respiratory symptoms at the out-set. About 10 percent to 20 percent of patients have diarrhea. After 2 to 7 days, SARS patients may develop a dry cough. Most patients develop pneumonia. The main way that SARS seems to spread is by close person-to-person contact. The virus that causes SARS is thought to be transmitted most readily by respiratory droplets (drop-let spread) produced when an infected person coughs or sneezes. The SARS NAT Test System for amplification and detection of the SARS coronavirus genome includes well preserved primers and ready-to-use, standardized reagents.The amplification product, obtained from a double amplification us-ing the nested method, is a 109 bp fragment and it is detected by gel electrophoresis.


AMS91 SARS qualitative kit 24 test

��complete kits / human diagnostics / bacteria

mycobacterium tuberculosis (MTB)This test shows up the presence of Mycobacterium tuberculosis, which causes tuberculosis.After a steady decline of cases in developing countries, the inci-dence of this pathology has recently increased, particularly in indi-viduals whose reduced immune defence facilitates the appearance of extremely serious clinical forms resistant to antibiotics (about 10% of AIDS patients present forms of tuberculosis).Classical laboratory diagnosis, the only one that can detect infec-tion with absolute certainty and one which is still based on culture tests to isolate the bacterium, takes a long time to produce results.Diagnosis based on PCR analysis has the advantage of allowing rapid intervention and, if necessary, the isolation of the patient to prevent the spreading of infection and to administer the appropriate pharmacological therapy.The kit uses a set of primers that specifically amplify a sequence within the IS6110 region.


AMS19 M. tuberculosis IS6110 region qualitative kit 24 test

AMS19/F M. tuberculosis IS6110 region qualitative kit 96 test

bacteria


helicobacter pylori (HP)Those tests show up the presence of Helicobacter pylori (HP) and the HP citotoxin-associated antigen ( cag-A) gene.The bacterium HP is a fastidious, microaerophilic spiral gram-nega-tive microorganism which plays a significant role in the pathologies of chronic gastritis, peptic ulcer and gastric cancer.Its worldwide distribution and high level of prevalence and the im-portance of associated pathologies make the elimination of HP a very useful approach to treating and controlling these gastroduode-nal diseases, since the eradication results in a marked reduction in the rate of recurrence of duodenal and gastric ulcer.The cag-A gene is an HP gene that Codes for a 96 to 138 kDa pro-tein that is associated with the production of citotoxic protein. The presence of HP cag-A gene positive strains is associated with pep-tic ulcer disease and gastric cancer.The Clonit’s PCR kits for amplification and detection of HP species-specific protein antigen sequence, and HP cag-A gene, includes well preserved primers, ready to use standardized reagents and pre-cast gels.


AMS20 Helicobacter pylori qualitative kit 24 test

AMS21 Helicobacter pylori cag-A gene qualitative kit 24 test


chlamydia trachomatisThis test shows up the presence of Chlamydia trachomatis, an obli-gate intracellular bacterium that represents the main case of sexu-ally transmitted diseases.C. trachomatis causes a variety of clinical syndromes in males (in-cluding urethritis and epididymitis), females (including cervicitis), and new borns (including conjunctivitis and pneumonia).Untreated or undiagnosed cervical infections in females can as-cend into the upper genital tract, causing pelvic inflammatory dis-ease and ectopic pregnancy.Infertility in males and females can result from untreated or undiag-nosed chlamydial infections. Cell culture is generally considered to be only 70 to 80% sensitive, it is labor intensive and requires 2 to 3 days for results.The PCR kit is a complete and rapid system for C. trachomatis nu-cleic acid extraction, amplification and detection.The kit uses a set of primers that specifically amplify a sequence of the CrP gene (cystein-rich protein).


AMS22 Chlamydia trachomatis qualitative kit 24 test


chlamydia pneumoniaeThis test shows up the presence of Chlamydia pneumoniae, a major cause of acute respiratory tract disease in humans and responsible for both endemic and epidemic pneumonia.In addition this organism has been associated with other clinical mani-festations, including coronary artery disease, asthma and sarcoidosis.The finding that 50 to 60% of adults in the diverse geographical places studied have serological evidence of C. pneumoniae infec-tion makes this one of the most prevalent infectious agents.Because the C. pneumoniae immunoglobulin G (IgG) antibodies persist for long time periods and, in reinfection, IgM or complement fixation antibody is often absent, differentiation of reinfection from previous infection is difficult.The difficulty in isolating and/or demonstrating the presence of the organism has made epidemiological studies of transmission, host range, tissue tropism and associations of the organism with specific clinical manifestations more difficult.The PCR kit based on nested amplification is a very powerful diag-nostic method, with great sensitivity and extremely rapid.A nested primer pair is used to specifically amplify a sequence of the RNA ß polimerase gene.


AMS25 Chlamydia pneumoniae (“nested”)



mycoplasma pneumoniae This test shows up the presence of Mycoplasma pneumoniae.Mycoplasmas (Mycoplasmata) are small (0.2 to 0.3 _m) pleomor-phic organism bounded only by a cell membrane with no evidence of a cell wall.M. pneumoniae is responsible for a wide spectrum of respiratory diseases. Most of them are mild infections, however this pathogen is an important cause of primary atypical pneumonia and accounts for as much as 10% of total X-ray proven pneumonia.In contrast to most other respiratory infections, M. pneumoniae in-fections are not seasonal and may be found as frequently in sum-mer as in winter. In large populations the outbreaks may appear as local epidemics.The disease spreads slowly in families and transmission apparently requires close contact.The cultivation of M. pneumoniae is time-consuming and may re-quire 3 weeks for results. Serological procedures are the most wide-ly used and require the demonstration of a rise in antibody titer. It takes too long for results of both of these diagnostic methods to be obtained toallow for the rapid application of an effective treatment.Clonit’s PCR nested kit is a rapid and highly specific and sensi-tive procedure to replace the culture method for detecting M. pneu-moniae. A nested primer pair is used to amplify a sequence of the D02 gene.


AMS26 Mycoplasma pneumoniae (“nested”)


�0complete kits / human diagnostics / bacteria

legionella pneumophilaThis test shows up the presence of Legionella pneumophila, a pri-marily aquatic saprophyte.The major reserve of Legionella spp. appears to be fresh-water sites, air-conditioning units and various drinking-water-plumbing fixtures.L. pneumophila is the agent of legionnaires disease, a multisystem disease manifested primarily as pneumonia.The clinical importance of legionnaires disease is twofold: (i) antimi-crobial agents that are usually used to treat pneumonia are ineffica-cious against legionellosis; (ii) the disease can occur in epidemic form, with a high fatality rate in untreated, and it is being increasingly recognised as a major cause of nosocomial pneumonia.This sensitive detection system is based on amplification of a chro-mosomal DNA sequence from L. pneumophila by Polimerase Chain Reaction.Clonit’s nested kit is based on a double amplification of a sequence within the macrophage infectivity potentiator (mip) gene.


AMS27 Legionella pneumophila (“nested”)


�1complete kits / human diagnostics / bacteria

borrelia burgdorferiThis test shows up the presence of Borrelia burgdorferi.Lyme Borreliosis (LB), caused by the spirochete B. burgdorferi, is the most common tick-borne disease in Europe as well as in U.S.A.In nature, this spirochete is primarily transmitted among wild ani-mals by infected ticks (in Europe by Ixodes ricinus).Infection in humans is coincidental and can be manifested with a broad spectrum of clinical signs. Symptoms of the disease vary from subclinical infections to severe skin, joint and neurological manifesta-tions. Diagnosis of LB relies mainly on clinical findings and serological tests. Antibodies against B.burgdorferi are not detectable in all cases of LB. Isolation of the spirochete from different patients specimens is difficult and requires special procedures. PCR makes the microbio-logical diagnosis of B. burgdorferi infection rapid and sensitive.Clonit’s PCR kit uses a set of primers that amplify a sequence of the flagellin gene.


AMS29 Borrelia burgdorferi qualitative kit 24 test


periodontal diseaseDestructive periodontal diseases are infections diseases character-ised by inflammatory changes in the periodontal tissues leading to periodontal attachment and alveolar bone destruction.It is widely accepted that the disease occurs as a result of infec-tion from subgingival plaque bacteria, particularly Gram-negative anaerobes. The identification of specific periodontal pathogens by conventional methods, mainly anaerobic cultivation, is difficult, time consuming and even sometimes unreliable.Therefore, a multiplex PCR kit for simultaneous detection of Acti-nobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia makes the identification of these specific bac-teria rapid and easy.The PCR multiplex kit uses sets of primers that amplify sequences within the rRNA 16S gene.


AMS65 Periodontal Disease Multiplexed qualitative kit 24 test


salmonella (extraction from stool)This test shows up the presence of S. agona, anatum, arizona, berta, bovismorbificans, brandenburg, brednel, broughton, corvallis, derby, dublin, dusseldorf, enteritidis, escanaba, gallinarum, give, hadar, hei-delberg, infantis, istanbul, javiana, jerusalem, johannesburg, livings-ton, london, maiduguri, muenster, ohio, saintpaul, stockholm, typhi, typhimurium e westerstede.Members of the genus Salmonella are Gram-Negative, facultative anaerobic, intracellular Bacteria that invade the mucous membrane and are spread primarily via fecal-oral transmission.There are more than 2000 different Salmonella serotypes and most of these serotypes are human pathogens.Foods of animal origin, such as milk and poultry, are frequently implicated in human infection because of the high prevalence of Salmonella Strains in domestic animals. Salmonellosis accounts for over 30 million cases of food-born infections annually, and can in-cur tremendous economical cost from lost productivity and medical treatment .In stool specimens or food products, potentially pathogenic bacte-ria may be present in low numbers, requiring their isolation by time. Consuming and labor-intensive culture techniques.The PCR technique provides a new strategy for rapid and sensitive detection of Salmonella pathogens.The kit use a set of primers that amplify a sequence of the invA gene.


AMS64 Salmonella qualitative kit 24 test


listeria monocytogenes (extraction from stool)This test shows up the presence of Listeria monocytogenes. Oligonucleotide primers were chosen to amplify a region of hlyA gene spanning a conserved HindIII site.L. monocytogenes is a Gram-positive facultative anaerobe micro-organism, that can grow over a wide temperature range. It is an important pathogen associated with abortion and encephalitis not only in sheep and cattle, but also in human.In pregnant women this organism often causes an influenza-like bacteremic illness that, if untreated, may lead to amnionitis and in-fection of the fetus, resulting in abortion, or premature birth.In some regions L. monocytogenes is the second most common cause of neonatal meningitis and the second most common single cause of bacterial meningitis in over 60 persons. Various reports show that L. monocytogenes can occur in dairy products and meat, faecal carriagein healthy individuals is common.The haemolysins elaborated from L. monocytogenes represents the virulence factor in the pathogenesis of listeriosis.


AMS28 Listeria monocytogenes qualitative kit 24 test


escherichia coli O1��:H� (EHEC/EPEC)(extraction from stool)This test shows up the presence of Enterohaemorrhagic and Entero-pathogenic (EPEC) E. coli.E. coli is the major constituent of the human aerobic fecal flora; some E. coli also represents one of the most important bacterial pathogens in medical and alimentary bacteriology, being identified as the causative agent of intestinal or urinary tract infections. Such strains produce additional factors responsible for their pathogenic-ity which are called virulence factors.One of these factors produces “attaching and effacing” (A/E) lesions and is characterized by dissolution of the brush border membrane and loss of microvillus structures at sites of bacterial attachment.Among E. coli the A/E gene is the specific marker for EHEC and EPEC (O157:H7 too) and PCR kits are based on the amplification of this coding region.


AMS41 E. coli O157:H7 (EHEC/EPEC) qualitative kit 24 test


bacillus anthracisAnthrax is a disease caused by the spore-forming Bacillus anthracis, a Gram-positive, rodshaped bacterium. In humans, 3 types of an-thrax infection occur: inhalational, cutaneous, and gastrointestinal. Naturally occurring inhalational anthrax is now a real cause of hu-man disease.Cutaneous anthrax is the most common naturally occurring form, with an estimated 2000 cases reported annually. Gastrointestinal anthrax follows ingestion of insufficiently cooked contaminated food and in-cludes two distinct syndromes, oropharyngeal and abdominal.Inhalational anthrax is expected to account for most morbidity and essentially all mortality caused by the use of anthrax as a biological weapon. Inhalational anthrax begins with non specific symptoms of malaise, fever, myalgia and nonproductive cough. After a period of 2-3 days this is followed by a sudden onset of severe respiratory distress associated with diaphoresis, cyanosis and increased chest pain. Death follows in 24-36 hours from respiratory failure, sepsis and shock. The need for a rapid diagnosis of anthrax is critical for a successful treatment of patients and infection resolution. In particu-lar, rapid detection in nasal swabs of spore inhalated is very useful to address a prophylactic antibiotic treatment.New diagnostic techniques are based on the use of the polymerase chain reaction to amplify markers specific to Bacillus anthracis. These new rapid methods may become useful in the clinical setting, where early diagnosis is crucial.Clonit’s kit is a rapid test able to detect the presence of Bacillus anthracis in clinical samples within 4 hours. The kit is based on the detection of capA (encapsulation protein gene) and pag (protective antigen gene) genes.


AMS69 Bacillus anthracis qualitative kit 24 test

��complete kits / human diagnostics / protozoa

plasmodiumThese tests show up the presence of Plasmodium genere, and of the four plasmodia that infect humans: P. falciparum, P. ovale, P. ma-lariae, P. vivax. Malaria most commonly presents itself as an acute systemic, febrile illness but may manifest more indolently as chronic anaemia, glomerulonephrites, or tropical splenomegaly syndrome due to this four plasmodia. Infections caused by P. falciparum are more fulminant than those caused by other plasmodia and may produce coma and renal fail-ure within 2 to 3 days in non immune patients. Malaria is transmitted to humans through the inoculation of infectious sporozoites by fe-male anopheline mosquitoes. For currently ill patients, the infecting species is usually either P. falciparum or P. vivax and can be identi-fied microscopically by using criteria of cell size, variety of parasite stages and multiply infected erythrocytes.In many cases it may be difficult or impossible to distinguish be-tween P. Falciparum and P. vivax or P. ovale and P. malariae. In such situations and in patients who may be infected by more than one malaria species, PCR kits represent a subtle, extremely sensitive and rapid method. Identification is based on a hypervariable region within the 18S rRNA gene.


AMS66 Plasmodium screening qualitative kit 24 test

AMS42 Plasmodium falciparum qualitative kit 24 test

AMS43 Plasmodium ovale qualitative kit 24 test

AMS44 Plasmodium malariae qualitative kit 24 test

AMS45 Plasmodium vivax qualitative kit 24 test

protozoa


leishmaniaThis test shows up the presence of Leishmania spp. that are the cause of leishmaniasis.Leishmania spp. parasitize monocytes and macrophages and in hu-mans lives as obligate intracellular parasites.Some are able to proliferate at a temperature of 37°C and cause visceral leishmaniasis; others prefer lower temperatures and cause coetaneous or mucocoetaneous leishmaniasis. As with many infec-tions, host factors are important and immunocompromised people such as those with acquired immunodeficiency syndrome (AIDS) may develop a more severe form of the disease.The diagnosis is usually based on morphologic diagnosis or culture from blood, aspirated or skin scrapings in the coetaneous forms of the disease.Culture technique may provide false negative results if antibiotics are not added to the culture to suppress bacterial growth or if the biopsy or scrapings are inadequate.The Leishmania spp. PCR nested kit provides results within a day and the nested amplification makes this test highly sensitive and accurate.A nested primer pair is used to specifically amplify a sequence of the 18S rRNA region.


AMS63 Leishmania (“nested”) qualitative kit 24 test


toxoplasma gondiiThis test shows up the presence of Toxoplasma gondii.T.gondii is an obligate intracellular parasite, but culture attempts are frequently negative even in known cases.Infection may cause a variety of manifestations. Congenital infec-tion acquired in utero from mothers with acute infection may cause a devastating syndrome to the central nervous system and ocular abnormalities which may be fatal.Humans and a variety of other animals serve as an intermediate hosts for T. gondii, humans may acquire the infections by the ingestion ei-ther of oocysts in material contaminated with cat faeces or of inade-quately cooked meat containing cysts from other intermediate hosts.A nested primer pair is used to specifically amplify a sequence of the B1 region.


AMS15 Toxoplasma gondii (“nested”) qualitative kit 24 test

AMS15/F Toxoplasma gondii (“nested”) qualitative kit 96 test

�0complete kits / human diagnostics / genetic

factor v leidenThe Factor V Leiden mutation is the most common clotting abnor-mality in patients with venous thromboembolism.In 1993 it was observed that plasma samples from certain individu-als with inherited thrombophilia were resistant to the anticoagulant action of activated protein C (APC), the protease generated by the thrombomodulin-PC anticoagulant pathway to inactivate activated factor V and VIII.APC resistance is associated with a point mutation in the Factor V gene that causes a hypercoagulable state by slowing the inactiva-tion of factor Va by APC.APC resistance accounts for 21% of deep-vein thromboses in the under-70s, and for up to 50% of familiar venous thromboses. More than 95% of cases of APC resistance are caused by Factor Leiden mutation.Clonit’s PCR kit shows up the presence of the mutation in heterozy-gous or homozygous form.


AMS50 Factor V Leiden 24 test

genetic

�1complete kits / human diagnostics / genetic

factor II (�0�10 GA variant)Since the discovery of the factor V Leiden mutation as the most common inherited disorder to venous thrombophilia and its appar-ent cosegregation with other well-established inherited prothrom-botic risk factors, evidence is accumulating that the association of double or multiple haemostatic defects greatly increase the pen-etrance of thrombotic disease.A genetic variation in the 3’-untraslated region of the factor II gene (G to A transition at nucleotide 20210) has recently been linked to in-creased plasma prothrombin levels and an enhanced risk of venous thrombosis. The high frequency of additional carriership for factor II20210 GA found in thrombophilic patients with the FV Leiden muta-tions indicates that the 20210A allele is an important additional risk for venous thromboembolism and might contribute to the thrombo-embolic manifestations.Clonit’s PCR kit performs factor II genotyping, it shows up the pres-ence of the mutation in heterozygous or homozygous form.


AMS51 Factor II 24 test

��complete kits / human diagnostics / genetic

MTHFR (�� CT variant)Hypermocysteinaemia has been shown to be a risk factor for coro-nary artery disease and venous thromboembolic disease.The methylenetetrahydrofolate reductase (MTHFR) 677 CT mutation has been described as a genetic risk factor that interacts with envi-romental factors such as a defiency of folic acid to cause Hyperho-mocysteinaemia.The mutation causes an amino acid substitution which renders the MTHFR protein thermolabile and, in the homozygous state, predis-poses to Hyperhomocysteinaemia.Clonit’s PCR kit shows up the presence of the mutation in heterozy-gous or homozygous form.


AMS52 MTHFR 24 test

��complete kits / human diagnostics / genetic

melanomaThe traditional approach used to study tumor markers has been based on the detection of substances that are either induced by or released by cancer cells.The Polymerase Chain Reaction has provided a means of detecting molecular markers present in low copy numbers with a significantly higher sensitivity than that typically achieved using antibody-based techniques.However, tissue-specific molecular markers may reflect the pres-ence of actual tumour cells in the circulation instead of tumor- as-sociated substances.Malignant melanoma cells can be detected with high sensitivity in the peripheral blood of patients using reverse transcription-PCR.Clonit’s nested PCR kits are based on the detection of tyrosinase mRNA; this mRNA is actively expressed only in melanocytes and melanoma cells indicates the presence of melanoma cells in pe-ripheral blood.


AMS70 Melanoma (RT + “nested”) qualitative kit 24 test

oncology

��

food diagnostics

��complete kits / food diagnostics

Genetically Modified Organisms (GMO)

BacteriaListeria monocytogenesE. coli O157:H7SalmonellaBacillus cereus

46

47

��complete kits / food diagnostics / gmo

genetically modified organisms (GMO)Clonit’s GMO kit is a screening method that shows up the presence of genetically modified organism.For a PCR screening method to be widely applicable, the following criteria should be met:

Ω primers should be selected that are specific for genetic elements present in a large number of genetically engineered agricultural crops

Ω the genetic elements on which the assay is based should not occur naturally in the respective plants

Ω the assay should not rely on genetic elements that occur in organisms that may appear frequently as contaminants of the foodstuffs under analysis.Clonit’s GMO kit follows these criteria.The kit is based on the detection of P-35S and NOS. The P-35S, promoter from the cauliflower mosaic virus, is the most abundantly used transgenic element in approved genetically engineered crops. The NOS, originally derived from Agrobacterium tumefaciens, is the most frequently used terminator in approved transgenic crops.The combination P-35S/NOS permits screening for approved ge-netically modified crops: corn, cotton, oilseed, rape, papaya, potato, tomato, soybean, tobacco, and chicory.


AMS80 GMO Kit qualitative kit 24 test

GMO

��complete kits / food diagnostics / bacteria

listeria monocytogenes (extraction from food)This test shows up the presence of Listeria monocytogenes in food.Oligonucleotide primers were chosen to amplify a region of hlyA gene spanning a conserved HindIII site.L. monocytogenes is a Gram-positive facultative anaerobe micro-organism, that can grow over a wide temperature range. It is an important pathogen associated with abortion and encephalitis not only in sheep and cattle, but also in human.In some regions L. monocytogenes is the second most common cause of neonatal meningitis and the second most common single cause of bacterial meningitis in over 60 persons.Various reports show that L. monocytogenes can occur in dairy prod-ucts and meat, faecal carriage in healthy individuals is common.The haemolysins elaborated from L. monocytogenes represents the virulence factor in the pathogenesis of listeriosis.The enrichment step we suggest and the PCR amplification allow identification of the organism in less than 30 h, while others meth-ods require a pure culture and are time consuming, or they require large numbers of target cells for positive identifications amongst non-target background organisms.

bacteria


AMS81 Listeria monocytogenes qualitative kit 24 test


escherichia coli O1��:H� (EHEC/EPEC)(extraction from food)This test shows up the presence of Enterohaemorrhagic and Entero-pathogenic (EPEC) E. coli.E. coli is the major constituent of the human aerobic fecal flora; some E. coli also represents one of the most important bacterial pathogens in medical and alimentary bacteriology, being identified as the causative agent of intestinal or urinary tract infections. Such strains produce additional factors responsible for their pathogenic-ity which are called virulence factors.One of these factors produces “attaching and effacing” (A/E) lesions and is characterized by dissolution of the brush border membrane and loss of microvillus structures at sites of bacterial attachment.Among E. coli the A/E gene is the specific marker for EHEC and EPEC (O157:H7 too) and PCR kits are based on the amplification of this coding region.


AMS82 E. coli O157:H7 (EHEC/EPEC) qualitative kit 24 test


salmonella (extraction from food)This test shows up the presence of S. agona, anatum, arizona, berta, bovismorbificans, brandenburg, brednel, broughton, corvallis, derby, dublin, dusseldorf, enteritidis, escanaba, gallinarum, give, hadar, hei-delberg, infantis, istanbul, javiana, jerusalem, johannesburg, livings-ton, london, maiduguri, muenster, ohio, saintpaul, stockholm, typhi, typhimurium e westerstede.Members of the genus Salmonella are Gram-Negative, facultative anaerobic, intracellular Bacteria that invade the mucous membrane and are spread primarily via fecal-oral transmission. There are more than 2000 different Salmonella serotypes and most of these sero-types are human pathogens.Foods of animal origin, such as milk and poultry, are frequently implicated in human infection because of the high prevalence of Salmonella Strains in domestic animals. Salmonellosis accounts for over 30 million cases of food-born infections annually, and can in-cur tremendous economical cost from lost productivity and medical treatment .In stool specimens or food products, potentially pathogenic bacte-ria may be present in low numbers, requiring their isolation by time. Consuming and labor-intensive culture techniques.The PCR technique provides a new strategy for rapid and sensitive detection of Salmonella pathogens.The kit use a set of primers that amplify a sequence of the invA gene.


AMS83 Salmonella qualitative kit 24 test

�0complete kits / food diagnostics / bacteria

bacillus cereus (extraction from food)Bacillus cereus is one of the most important food pathogens. It may cause two distinct syndromes, i.e. diarrhoea and emesis, and be re-sponsible for some non-gastrointestinal infections.This organism is often isolated from food samples, such as raw milk, dairy products and rice products, as a contaminant. It is a spore-form-ing bacterium and is widespread in the environment. It has been re-ported that there are several different toxins produced by Bacillus ce-reus: enterotoxins, haemolysin, phospholipase C and emetic toxin.Clonit’s kit is a rapid test able to detect the presence of Bacillus ce-reus in food samples. The enrichment step we suggest and the PCR amplification allow identification of this organism in less than one day. On the contrary other methods are time consuming and require a pure culture, or large numbers of target cells for positive identifications amongst non-target background organisms.The kit use a set of primers that amplify a sequence of the 16S rDNA.


AMS62 Bacillus cereus qualitative kit 24 test

�1

veterinary diagnostics

��complete kits / veterinary diagnostics / virus

feline infectious peritonitis virus (FIPV)Feline Infectious Peritonitis (FIP) is caused by a coronavirus that can infect any cat, though young cats and very old cats (14yr and up) appear most susceptible. The FIP virus (FIPV) is very similar to the coronavirus that causes a transient, usually mild, self-limiting diarrhea (Feline Enteric Corona Virus, FECV).The type and development of disease is quite complex and, in large part, dependent on the status of the animal’s immune system. The most common clinical signs are non-specific and include fluctuating fever, inappetance, lethargy and weight loss. Sometimes, if the central nervous system is affected, neurological abnormalities are apparent.The genetic material of FIPV is RNA. Once the virus infects a cat’s cell, it makes a DNA copy of its RNA genome and inserts this copy into the DNA of the host cell. DNA from feline cells can be extracted and tested to determine if any of these cells have been succesfully infect-ed with FIPV. Using PCR kit it is possibile to make millions of copies of the sequences specific to FIPV, and then visually detect the PCR products. PCR-base testing is extremely sensitive and highly specific, markedly reducing the chance of false positive and negative results.

virus


AMS48 FIPV (RT + “nested”) qualitative kit 24 test

��

single components

��single components

Extraction KitRNA extraction from serum or plasma DNA extraction from blood DNA extraction from expectorate DNA extrac.from expct.with Phenol-Cl DNA Isolation System (small sample volume)DNA Isolation System (large sample volume)

Master Mix

Positive Control

55

56

58

��

extraction kitClonit’s extraction kits allow isolation of genomic and viral DNA/RNA from whole blood, plasma, serum, expectorate and, enviromental sources.


EX01 RNA extraction from serum or plasma 50 extr.

EX02 DNA extraction from blood 50 extr.

EX03 DNA extraction from expectorate 50 extr.

EX03/F DNA extrac. from expct. with Phenol-Cl 50 extr.

EX11 DNA Isolation System (small sample volume) 50 extr.

EX12 DNA Isolation System (large sample volume) 50 extr.

single components

��

master mixThe master mix is the “heart” of all clonit’ kits and contain all the necessary components to perform PCR reaction with the exception of Taq Polymerase. The master mixes, togheter with the posive con-trols, are useful for routine diagnostic laboratories where is placed an indipendent extraction station. All DNAs and RNAs of PCR quality can be used with Clonit master mixes.The PCR master mix has been optimized for use in routine PCR for amplifying DNA/RNA templates.Amplification solution is stable for 12 months at –20°C.


AMS01-2 HBsAg gene 24

AMS02-2 HBsAg gene 24

AMS03-2 HIV DNA gag gene (“nested”) 24

AMS04-2 HIV DNA env gene (“nested”) 24

AMS05-2 HTLV-I 24

AMS06-2 HTLV-II 24

AMS07-2 HPV total 24

AMS08-2 HPV 16 24

AMS09-2 HPV 18 24

AMS10-2 HPV 6 24

AMS11-2 HPV 11 24

AMS12-2 CMV (“nested”) 24

AMS13-2 EBV (“nested”) 24

AMS14-2 HCV - RNA 5’UTR (RT+”nested”) 24

AMS15-2 Toxoplasma (“nested”) 24

AMS16-2 HBcAg precore mutation 24

AMS17-2 Parvovirus B19 24

AMS18-2 HSV 1 and 2 24

AMS19-2 Mycobacterium tuberculosis 24

AMS20-2 Helicobacter pylori 24

AMS21-2 Helicobacter pylori CagA gene 24

AMS22-2 Chlamydia trachomatis 24

AMS23-2 VZV 24

AMS24-2 JCV 24

AMS25-2 Chlamydia pneumoniae 24


AMS01-2/F HBsAg gene 96

AMS02-2/F HBsAg gene 96

AMS03-2/F HIV DNA gag gene (“nested”) 96

AMS04-2/F HIV DNA env gene (“nested”) 96

AMS05-2/F HTLV-I 96

AMS06-2/F HTLV-II 96

AMS07-2/F HPV total 96

AMS08-2/F HPV 16 96

AMS09-2/F HPV 18 96

AMS10-2/F HPV 6 96

AMS11-2/F HPV 11 96

AMS12-2/F CMV (“nested”) 96

AMS13-2/F EBV (“nested”) 96

AMS14-2/F HCV - RNA 5’UTR (RT+”nested”) 96

AMS15-2/F Toxoplasma (“nested”) 96

AMS16-2/F HBcAg precore mutation 96

AMS17-2/F Parvovirus B19 96

AMS18-2/F HSV 1 and 2 96

AMS19-2/F Mycobacterium tuberculosis 96

AMS20-2/F Helicobacter pylori 96

AMS21-2/F Helicobacter pylori CagA gene 96

AMS22-2/F Chlamydia trachomatis 96

AMS23-2/F VZV 96

AMS24-2/F JCV 96

AMS25-2/F Chlamydia pneumoniae 96

single components

��


AMS26-2 Mycoplasma pneumoniae 24

AMS27-2 Legionella pneumophila 24

AMS28-2 Listeria monocytogenes 24

AMS29-2 Borrelia burgdorferi 24

AMS40-2 HPV 33 24

AMS41-2 E. coli O157:H7 (EHEC/EPEC) 24

AMS42-2 Plasmodium falciparum 24

AMS43-2 Plasmodium ovale 24

AMS44-2 Plasmodium malariae 24

AMS45-2 Plasmodium vivax 24

AMS46-2 Poliovirus (RT+Single amplification) 24

AMS47-2 HIV RNA gag gene (RT+”nested”) 24

AMS48-2 FIPV (RT+”nested”) 24

AMS50-2 Fattore V Leiden 24

AMS51-2 Fattore II (G20210A) 24

AMS52-2 MTHFR (C677T) 24

AMS62-2 Bacillus cereus 24

AMS63-2 Leishmania 24

AMS64-2 Salmonella 24

AMS65-2 Peridontal disease multiplex 24

AMS66-2 Plasmodium screening 24

AMS69-2 Bacillus anthracis 24

AMS70-2 Melanoma (RT+”nested”) 24

AMS80-2 GMO 24

AMS81-2 Listeria monocytogenes 24

AMS82-2 E. coli O157:H7 (EHEC/EPEC) 24

AMS83-2 Salmonella 24

AMS91-2 SARS 24

AMS92-2 HBsAg gene “nested” 24


AMS26-2/F Mycoplasma pneumoniae 96

AMS27-2/F Legionella pneumophila 96

AMS28-2/F Listeria monocytogenes 96

AMS29-2/F Borrelia burgdorferi 96

AMS40-2/F HPV 33 96

AMS41-2/F E. coli O157:H7 (EHEC/EPEC) 96

AMS42-2/F Plasmodium falciparum 96

AMS43-2/F Plasmodium ovale 96

AMS44-2/F Plasmodium malariae 96

AMS45-2/F Plasmodium vivax 96

AMS46-2/F Poliovirus (RT+Single amplification) 96

AMS47-2/F HIV RNA gag gene (RT+”nested”) 96

AMS48-2/F FIPV (RT+”nested”) 96

AMS50-2/F Fattore V Leiden 96

AMS51-2/F Fattore II (G20210A) 96

AMS52-2/F MTHFR (C677T) 96

AMS62-2/F Bacillus cereus 96

AMS63-2/F Leishmania 96

AMS64-2/F Salmonella 96

AMS65-2/F Peridontal disease multiplex 96

AMS66-2/F Plasmodium screening 96

AMS69-2/F Bacillus anthracis 96

AMS70-2/F Melanoma (RT+”nested”) 96

AMS81-2/F Listeria monocytogenes 96

AMS82-2/F E. coli O157:H7 (EHEC/EPEC) 96

AMS83-2F Salmonella 96

AMS91-2/F SARS 96

AMS92-2/F HBsAg gene “nested” 96

single components

��

positive controlEach PCR assay need a control! These controls are useful for routine diagnostic labs where are placed indipendent extraction units. They can be used in association with the corrisponding master mixes. The positive control can be useful to the evaluation of “home brew” systems once sure thet the primers fit the cloned region.

Code Product description Q. ty

AMS01-3 HBV (HBsAg gene) 50 ng

AMS02-3 HBV (HBcAg gene) 50 ng

AMS03-3 HIV (gag gene) 50 ng

AMS04-3 HIV (env gene) 50 ng

AMS05-3 HTLV-I 50 ng

AMS06-3 HTLV-II 50 ng

AMS07-3 HPV total 50 ng

AMS08-3 HPV 16 50 ng


AMS10-3 HPV 6 50 ng


AMS12-3 CMV 50 ng

AMS13-3 EBV 50 ng

AMS14-3 HCV 50 ng

AMS15-3 Toxoplasma 50 ng

AMS16-3 HBcAg precore mutation 50 ng

AMS17-3 Parvovirus B19 50 ng

AMS18-3 HSV 1 and 2 50 ng

AMS19-3 Mycobacterium tuberculosis 50 ng

AMS20-3 Helicobacter pylori 50 ng

AMS21-3 Helicobacter pylori cagA gene 50 ng

AMS22-3 Chlamydia trachomatis 50 ng

AMS23-3 VZV 50 ng

AMS24-3 JCV 50 ng

AMS25-3 Chlamydia pneumoniae 50 ng

AMS26-3 Mycoplasma pneumoniae 50 ng

AMS27-3 Legionella pneumophila 50 ng

AMS28-3 Listeria monocytogenes 50 ng

single components

��

Code Product description Q. ty

AMS29-3 Borrelia burgdorferi 50 ng


AMS41-3 E. coli O157:H7 50 ng

AMS42-3 P. falciparum 50 ng

AMS43-3 P. ovale 50 ng

AMS44-3 P. malariae 50 ng

AMS45-3 P. vivax 50 ng

AMS46-3 Poliovirus 50 ng

AMS47-3 HIV gag 50 ng

AMS48-3 FIPV 50 ng

AMS50-3 Factor V 50 ng

AMS51-3 Factor II 50 ng

AMS52-3 MTHFR 50 ng

AMS62-3 B. cereus 50 ng

AMS63-3 Leishmania 50 ng

AMS64-3 Salmonella 50 ng

AMS65-3 Peridontal disease multiplex 50 ng

AMS69-3 B. anthracis 50 ng

AMS70-3 Melanoma 50 ng

AMS80-3 GMO 50 ng

AMS91-3 SARS 50 ng

single components

�0

molecular diagnosis

�1molecular diagnosis

automated DNA sequencertrombophilia kit

real time PCRquantitative kit hepatitis b virus (HBV) hepatitis C virus (HCV) Toxoplasma gondiiallelic discrimination factor V leiden factor II G20210A

62

63

��molecular diagnosis / automated dna sequencer

thrombophilia kit (factor V, factor II, MTHFR)Venous thrombotic events (VTE) are quite common. They affect ap-proximately one of every 1,000 people per year, cause 60,000 deaths annually, and have a lifetime clinical prevalence of approximately 5% (1,2). Until recently, the medical community did not regard thrombo-philia as genetic disease, but today, it is one of the best examples of a common disease for which specific genetic defects have been identified in several genes.Up to 60% of typical thrombophilic patients have one or several of the six major genetic alterations that have been linked to the dis-ease. And in the last six years, researchers have described three new common genes involved in thrombophilia: factor V; prothrom-bin; and MTHFR. Defects in these genes are now identified in more than half of all cases of inherited thrombophilia. The mutations in each of these genes are well conserved, single-nucleotide substi-tutions for which direct DNA-based assays are being increasingly used to determine thrombotic risk. In comparison, the more widely recognized defects in the anticoagulant proteins-antithrombin III, protein C, and protein S-are found collectively in less than 10% of all VTE patients Clonit’s Thrombophilia kit includes all the reagents for nucleic acid extraction, amplification of specific genomic regions, and simultaneous indentification of the three single-nucleotide sub-stitutions through fragment analysis on ABI Prism genetic analyser.


MD01 Thrombophilia kit qualitative kit 24

automatedDNA sequencer

��

quantitative kitThe PCR real time reaction exploits the 5’ nuclease activity of DNA Taq Polymerase to cleave a TaqMan® probe (TaqMan® probes are covered by US Patent 5723591 and foreign counterparts and pat-ents pending owned by Applera Corporation) during PCR. The Taq-Man® probe contains a reporter dye at the 5’ end of the probe and a quencher dye at the 3’ end of the probe. During the reaction, cleav-age of the probe separates the reporter dye and the quencher dye, which results in increased fluorescence of the reporter. Accumula-tion of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppres-sion of the reporter fluorescence primarily by Förster-type energy transfer. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites.The 5’ to 3’ nucleolytic activity of the DNA Taq Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then displaced from the target, and polymerization of the strand continues. The 3’ end of the probe is blocked to prevent extension of the probe dur-ing PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. The increase in fluo-rescence signal is detected only if the target sequence is comple-mentary to the probe and is amplified during PCR. Because of these requirements, non specific amplification is not detected.The following kits are based on the Taqman® technique.

hepatitis B virus (HBV)

real time PCR

molecular diagnosis / real time PCR


05960126 HBV-DNA Real Time Master Mix qualitative kit 96

hepatitis C virus (HCV)


05960219 HCV-RNA Real Time Master Mix qualitative kit 96

toxoplasma gondii


05960129 Toxoplasma Real Time Master Mix qualitative kit 96

��molecular diagnosis / real time PCR

the picture above shows

in order:

- standard diluition

- calibration curve

- results

��

allelic discriminationThe Allelic discrimination exploits the 5’ nuclease activity of DNA Taq Polymerase to cleave a Taqman MGB probes® (TaqMan® probes are covered by US Patent 5723591 and foreign counterparts and pat-ents pending owned by Applera Corporation) during PCR. The MGB probes® contains a reporter dye at the 5’ end of the probe and a no fluorescent quencer (NFQ) group at the 3’. Fluorogenic probes with the MGB (minor groove binder) attached to the 3’ end perform well in the 5’ nuclease assay. They are an improvement over unmodified probes because shorter sequences (13-to 20-mers) can be used to obtain probe that have an optimal Tm (65 to 67). Thus, attachment of the MGB enables the use of shorter fluorogenic probes, which results in improved mismatch discrimination.Mismatches between a probe and target reduce the efficiency of probe hybridizaton. Furhermore, Taq Polymerase is more likely to displace the mismatched probe rather than cleave to it, releasing the reporter dye. Open the pre- read allelic discrimination plate doc-ument. Click instrument tab and select the post read button. The result will show the pre-read data subtracted from the post-read data for the run. The following kits are based on the MGB Probes®.

factor V of leiden

molecular diagnosis / real time PCR


05960127 Factor V of Leiden 96

factor II G�0�10A


05960128 Factor II G20210A 96

PCR process is covered by Patent 4,683,195 and 4,683,202 (Euro-pean Patent n.201.184 and n.200.362) owned by HLR, Inc. Use of the process may require a licence

��molecular diagnosis / real time PCR

the picture above shows the

allelic discrimination test

��

CLONpactCLONpact is an innovative system highly suitable for separating by gel electrophoresis molecules of biological interest such as DNA.The system combines two different devices:

Ω CLONgel – a ready to use pre-cast agarose gel in UV transparent cassette with removable cover containing ethidium bromide in a 12-well format.

Ω CLONpact – an automatic device for CLONgel cassettes with integrated power supply and UV transilluminator.

Ω CLONpact offer a series of important advantages:

Quickness: time saver, no need to lose time for gel preparation.Standardization: each lot of CLONgel is cecked for quality standards at manufactoring stage.Band Collecting: top part of the cassette is removable and it gives change to easily collect the separated bands for any further prepa-ration or investigation.Easy to use: CLONpact system is complete and it does not need any other instrument or reagent to be used.Safety: the system allows electrophoresis runs without touching gel or adding buffer solution in respect to the operator safety and health.Cost saving: CLONpact offers several advantage at almost no extra charge if compared with standard agarose gels preparation.

CLONgel Pre cast agarose gel

lab equipments required but not supplied

Code 24 box pcs %Agarose (EtBr 0,5g/ml)

Resolution Range as base pairs

Well Volume Mean run time in minutes

Mean voltage supply

Shelf lifeat roomtemperature

CLG001 1% 250bp – 5kb 26-27μls 20-40 60-80V 12 months

CLG002 2% 100bp – 1kb 26-27μls 20-40 60-80V 12 months

CLG003 4% 20bp – 200bp 26-27μlf 20-40 60-80V 12 months

CLONpact

code Volts c.c Max current Programs store UV wavelenght Maxconsumption

Line voltage 50/60 Hz

CLP001 Up to 120 V 80 mA 10 312 nM 150 W 115-230 V.A.C.

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Documents

BIOLOGIA MOLECOLARE - CPM SAS · quality control in molecular biology The use of the Polymerase Chain Reaction (PCR) and the NAT tech-nologies in molecular diagnostic has increased