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LCDC REPORT
The polymerase chain reaction: An overview and development of diagnostic PCR protocols at the LCDC
NATIONAL LABORATORY OF ENTERIC PATHOGENS, BUREAU OF MICROBIOLOGY,
LABORATORY CENTRE FOR DISEASE CONTROL
THE DEVELOPME TIN TilE LATE 1980S OF A PIWPRIETARY
method for in vitro amplification of specific DNA or RNA sequences by lhe polymerase chain reaction (PCR) has revolutionized molecular biology. The PCR has many applications in biology and affords tremendous early diagnostic potential in many areas of medicine and infectious diseases. The key lo the success of the PCR in diagnosis resides in ils ability lo amplify regions within a single molecu le of DNA which may have etiologic significance.
The PCR can readily produce more than a million copies of a specific DNA or RNA sequence in a simple three-step cycling process. The initial step involves the denaturation of double-stranded DNA lo separate the complemenlmy strands . and lhe second step allows for the annealing of primers lo lhe dissociated DNA strands. Third. the primers participate in a n extension reaction catalyzed by a thermostable DNA polymerase. and the cycle is then repealed. The PCR reaction uses two primers complementary lo and hybridizing with opposite strands of Lhe DNA wilh one lo lhe lefl (5') and one to Lhe right (3') of the largel sequence lo be amplified . If lhe template is an RNA sequence. a DNA copy (eDNA) musl firsl be synthesized using a reverse lranscriplase before the PCR is initialed. The DNA copies or 'amplicons· are typically produced in a few hours after approximately 30 3 lo 5 min cycles in which enzymalic extension lheorelically doubles the amount of DNA from the previous cycle. Although different DNA polymerases have been used in PCR applications . lhe mosl convenient is the thermostable polymerase "Taq· isolated from Thennus aquaLicus. Taq polymerase is capable of with standing lhe high temperatures required to denature
DNA. and has permitted optional automation of lhe technique using programmable thermal cyclers now available from a variety of commercial sources.
The mosl common method of visualizing amplicons generated in Lhe PCR procedure is gel electrophoresis in polyac1ylamide or agarose. followed by elhid ium bromide staining. The observed sizes of the amplified fragment should be identical lo those predicted from the known nucleotide sequence. Nonspecific hybridization of the primers lo sequences other U1an those targeted usually generales amplification fragments of sizes different from lhe desired amplicon. Because such nonlargeted amplification is nol uncommon, methods such as ultraviolet spectroscopy and fluoromeliy. which only indicate an increase in lhe lola! amount of DNA present. shou ld be avoided as a means of detection. In order lo ensure lhal lhe tcu·gel sequence has been amplified, il is recommended lhal the specificity be tested by probing a Southern blot of lhe analytical gel. or dol-blots. with labelled probes nested between the PCR primers and representing a portion of the amplified sequence. or that restriction endonuclease digestions specifi c for sites within lhe amplicon should be used . For a ll PCR applications. sample preparation techniques and amplification and detection methods need lo be op timized during lhe research and developmental phase.
ll is recognized lhal DNA polymerases do not duplicate DNA with complete fidelity. and nucleotide misincorporalion does occur. In nature most of these polymerases possess a proofreading activity which will remove any misincorporaled nucleolides cu1d rep lace them with the correct base. Commercially prepared
Correspondence ancl reprints: Dr WM Johnson. Bureau qfMicrobio/ogy. Laboratory Centre for Disease Control. Tunney's Pasture. Ollawa. Ontario KIA OL2
CAN J INFECT DIS VOL 2 No 2 SUMMER 1991 89
LCDC report
polymerases often lack this proofread ing function. as is the case for Taq polym erase. a nd a base s ubsti tuti on error ra te of one in I 0.000 bases polymer·ized has bee n estima ted for Taq (l ). The potenti a l for nucleo tide mis incorporalion has necess itated modilka tions in es tabli shed techniques when sequencing data is to be obtained s ubseque nt to PCR a mplification . Maximum fide lity can be ach ieved by a combina tion of opti ma l in vitro deO>.J'nucleoside triphospha te concentra tions. pi I. divalent metal cations. ionic strength a nd tempera ture cond itions . Devia tions from these estab li s hed optima may result in a noma lous amplification products.
False positive ampli fi ca tions are a potential problem with a ll di agnostic PCR application s. and the most serious sou rce a ri ses from the carryover of DNA from a previous am plifica tion of the same target sequence rather than from sa mple-to -sa mple con tamina tion dlll·ing cont emporaneous processing. As a resu lt . preca u tions must be in place in the PCR laboratory to avo id thi s carryover. Essenti a l safeguards inclu de phys ical separation of pre- and pos t P R a mplifi ca tions . a li quoted reagents. positive di s pl acement pipelles. and judiciou s se lect ion of controls. in addition to meti c u lous technique when one is u s ing a variety of lab01·a tory equipment a nd supplies.
Fortunately. a wide varie ty of clin ical specimens is suitable for genotypic a na lys is in PCR a pplica tions. These specimens include whole blood or while blood cell s. other body Ouids s uch as urine or feces. c linica l swabs. dried smears a nd pa ra ffin -embedded tissues. Tissues fi xed in methanol or 50% e tha nol a re s uperio r to those fixed in forma lin in tha t the yield of DNA is higher a nd less degradation of nu cle ic ac ids occurs. Hepa rinized or citrated b lood has been used for DNA a na lys is . in a ddition to a ir-dri ed blood smears on s lid es optiona lly fixed with e thanol or metha nol.
Research a pplica tions of PC R technology a re numf' r ous. A pa rtial listing would include direc t genomic cloning of DNA or eDNA. gene ti c finge r-printing of foren s ic sa mples. the a na lys is of a ll e lic sequence variations. and direct nucleotide sequencing. The PCR has th e poten tia l to replace many conventiona l diagnostic tec h niqu es for infectiou s a nd gene tic diseases in clinica l med ic ine. T he PCR is currently be ing used to study genetic diseases such as hemophili a . cys tic fibros is. re tinoblastoma. Huntington's disease . s ickle cell a rJ e mia and beta-tha lassemia. von Willebra nd 's disease. Leber's op ti c neu ropa thy. muscula r dys trophy. phen y lketonuria. Tay-Sachs. a nd a lpha - 1-anlitryps in de fi c iency. The PCR can a lso be u eel to screen for point muta tions in the ins ulin gene. detect a single lym phoma ce ll in the presence of I 06 norma l cell s. s tudy chromosom a l trans locations. de tect growth hormon e gene de le tions . and determine huma n lymphocy te a nti gen (1-!LA) c lass II gene polymorphis m.
The PCR has a n advantage over the competing tech nology of DNA hybridiza tion in that the sensitivity is
90
su ffi c ient to a llow the direct detection of microbia l DNA in a high percentage of known positive pathologica l s pecimens. a qua lity not a lways found in DNA hybridi za tion methods. This genotypic technique, however. a lso de tects gene-harbouring stra ins, independent of ge ne expression. Hence, a positive result in the PCR is only indicative of the presence of Lhe targeted gene sequence a nd does not reOect the viability or pathogen ic toxic activities of the organism in the specimen. The PCR may supplement <irowth amplification protoco ls which can a llen fail to detect virulent s tra ins presen t at low levels in pathological or food samples. Frequently, nonpa thogenic strains of the same genu s or s pecies overgrow the pathogens, and strains m ay read ily lose plasmid- or phage-med iated virulence fac tors. The PCR al lows specific enzymatic r·eplication of targf' ted gene fi·agments and , s ince cell growth a nd rep li cation are not required , both injured a nd viable cell s wi ll be de tec ted a nd identifi ed with equal facility. In food s. a n indica tor of dead cell s yields va luable infornration as to the quality of the food , but may not be indica tive of a health hazard . The PCR can be perfo rmed using whole bacterial cell s without extraction of nucleic ac ids and, coupled with pre-en richment growth before the PCR, dilutes out DNA not being biologica lly dup lica ted, thus perm itting the identification of organisms in samples conta ining numbers of pathogenic ba ct eri a undetectab le by other routine m ethods. The icl entifkalion of target genes related i.o virulence by the PCR a lTe rs a very specific, sensitive, rela tive ly ra pid a nd inexpensive a lterna tive to tradi tional in vitro assays. which depend on adequate gene expression for re liab il ity and sens itivity. Th e interested reader is referred to more de tailed procedural informa tion in the la boratory manua ls (1 -3) a nd recent review a rticles on PCR (4- 18).
PCR application s for U1e d iagnosis of infectious disea es occu rred firs t with vira l infections, for which ea rly detection is particularly importa nt. PCR resu lts can be obta ined within one day of receipt or specimens in the la bora tory . There a re now specific a nd sensitive PCR protocols published for the de tec tion a nd typing or huma n genita l pa pillomaviruses, human immunodefi c iency viru s types l a nd 2, human T cell lymphotropic virus type l. hepatitis viruses A, B and C. severa l sc rotypes of huma n enteroviru ses. human herpes virus a nd rota viruses. huma n parvovirus B 19. rhinovirus , pseudora bies virus, rubella virus . pa ra myxoviruses ca u s ing mumps a nd measles, cytomegalovirus a nd Epstc in -Oa rr virus. The PCR has a lso been used to es ta bli s h the vira l e tiology in both enteroviral m eningitis and viral myoca rditis.
PCR procedures have been combined v,rilh probes ta rge ting Escherichia coli a ndlegionella gen es a nd have been a pplied to the detec tion or bacte ria l pathogens in environ men ta l water samples. This combina tion of techniques provides the necessary sensitivity and s pec ific ity required lor monitoring bac teria l pa thogens
CAN J INFECT DIS VOL 2 No 2 SUMMER 1991
in envi r onmental water . T o dale . PC R protocols have
also been published for acute typhus infec tion . d etec
tion of shigella in feces. toxigenic E coli. l o l a ! co l i form
bacteria. Leg ionella p neumophila . Mycobacterium spe
cies. Bordetella perll tss is. Mycoplasma pneumoniae.
Borrelia burgdOTferi. Trep onema pa llid um. Coxiella
burnettii. Cand ida a lbicans. Ricketts ia riclcet ts ii. Try panosoma congolense an d '[)·ypanosoma brucei subspe
cies. All of t hese in fectiou s d i ease can be d iagn o ed
with increased sensitiv i ty an d speci fic ity in a sh orter or
equal lime frame and at substantially less cost by PCR
t han by conventional techniques.
The application of PCR technology to th e del c tion
and diagnosis of bacteria l pathogens has been a recen t
p r iority at lhe Laboratory Centre for D isease Contro l
(LCDC). Dr D Russell Pollard from the National Labora
tory for Specia l Path ogens i n the Bureau of Microbiol
ogy was one of l h e first r esearcher in Canada to
develop PCR protocols to d etect pathogenic m icrobes
associated with hum an di sease . Since his prem a ture
and untimely death in J une of I 990. his co lleagu es
have continued the r evolu tionary work t ha t was initi
aled in his laboratory . Curren t r esear ch and develop
ment at the LCDC follows the ini tia tive establi shed by
D r Pollard and is focu sed on PCR d iagn os ti c p rotocols
targeting vi r u lence and pathogenic i ty fac tors assoc i
ated with a variety of in fectiou s agen ts im portant in
h u man d isease.
To dale. we have developed sever a l PCR protocols.
The first was for th e specific detection of Chla myd ia
REFERENC ES l. Erlich llA. Gibbs R. Kazazian 1111 Jr. Polymerase
chain reaction. In : Curren t Commu n ications in Molecular Biology. New York : Cold Spring Ha rbor Laboratory Press. 1989:6.
2. Innes MA. Gel fand DH. Sninsky JJ. White TJ . PCH Protocols. A Gu ide to Methods and Applicat ions. Sa n Diego: Academic Pr ess Inc. 1990 .
3. Sambrook J. FI-i tsch EF. Man iatis T. In v it ro ampl i fication of DNA by the polymcmse cha in reaction. In: Molecular Clon ing. A Laboratory Manual. 2nd edn. ew York: Cold Spring !!arbor Laboratory Press. 1989: 14.2-35.
4. Anderson R. Molecu lar considerations for the laboratory d iagno i of J apanese encephali tis vims. Southeast Asian J T rop Mcd Public llcal th 1989:20:605- 10 .
5. Eisenstein B l. The polym erase chain reac tion . A new method of using molecular genetics for medical d iagnosis. N Eng! J Mcd 1990:322: I 78-83.
6. Fox Rl. Dolan I. Com pton T. Fci liM. I lamer M . Saito I. Use of DNA amplification methods for clin ica l diagnosis in autoimmune d iseases. J Cli n Lab Anal 1989:3:378-87.
7. Goossens M. Recent developments in the d iagnos is of the hemoglobin d isorders. Nouv Rev Fr llcmatol 1990:32:63-5.
8. Grompe M. Gibbs RA. Chamberlain JS. askcy CT. Detection of new mutation disease in man and mou se. Mol Bioi Med 1989:6:51 I 2 I .
CAN J INFECT DIS VOL 2 No 2 SUMMER 199 1
LCDC report
species in bolh labora tory samples o f infected M cCoy
ce ll s and clin ical specimen s . In a collabora tive proj ect
between the LCD C and the Cad ham pr ovincial labor a
tory in M anitoba. para llel tes ting of th is PCR an d Abbott
Chlam y d iazyme"' procedures was r ecenlly completed
on 274 c linical specim en s w i th very p romisin g resu lts .
In other work. a ba ttery of verolox in-specific primers
has resulted in PCR protocols to speci fi cal ly detect and
dis tingu ish lhe genes for VTl. VT2. and VTe in human
and n onhuman iso lates of veroloxigenic E col i. One of
the PCR pro tocols d eveloped a t the LCDC in the ver a
toxin stu dy clearly distinguished lhe very close ly re
lated gen es coding for VT 2 and v r e. Additionally. a ve ry
significan t contribution focu sed on differ entia ti on of
the vi r tu a lly identica l gen es for the shigaloxin o f Shi
gella dysenter iae 1 and E coLi V rl. The verotox in PCR
pro tocols are currenlly b ing applied to lhe complete
gen otypic ana lysis of verot.oxigenic E coli isola ted from
re ta il m eats and ver a toxigenic E coLi associa ted with
pediatric h em olyti c uremic syndrom e.
T he LCD C has jus t publish ed a study on the detec
ti on o f l h e aerolysin gen e in clin ical iso la tes of
Aeromonas hy drophila by the PCR. and these p 1imers
should have application as a species-spec ific v iru len ce
p robe to dis tingui h bel a-h em oly tic stra ins of A hydro
p hi/a and Aeromonas sobri a. An o ther m aj or investiga
tion . a lso recenlly completed at the LCD C. describes the
detec ti on of gen es by the PC R for enterotoxins A to E ,
exfo l ia tive toxin s A and B . and toxic shock syndrome
toxin - ! in Staphy lococcus a ureus.
9. I Iusson RN. Com eau AM . Hoff R. Diagnosis of human immu nodeficiency virus infection in infan ts and child ren . Pediatrics I 990 :86: 1- 10.
I 0. J ackson J B. The polymerase chain reaction in trans fu ion m edicine. Transfu sion 1990:30:5 1-7.
I l . Kazazian HI I Jr. T he tha lassemia syndromes: Molecular basis and prenatal diagnosis in 1990. Semin llcmatol 1990 :27:209-28.
12. Macintyr e EA. T he use of the polymerase chain reaction in haematology. Blood Rev 1989:3:201 - 10.
13. Ra tner L. Vander Heyden N. Paine E. et al. Familial adult T -cell leukemia/ lymphoma. Am J Hem alol 1990:34:2 15-22.
14. Re iss J. Cooper DN . Applica tion of the polymerase chain reaction to the diagnosis of human genetic d isease. Hum Genet 1990:85: 1-8.
15. Rodu B. The polym erase chain reaction : The revolution within . Am J Med Sci 1990:299:2 10-6.
16. Rosenblatt JD. Zack JA, Chen IS. Lee H. Recent advances in detection of human T-cell leukemia viruses Lype I and type II infection. Nal lmmu n Cell Grov.rlh Rcgul 1990:9: 143-9.
17. Sninsky JJ. Kwok S. Detection of human immunodeficiency viruses by the polymerase chain reaction . Arch Pathol Lab Med 1990: 11 4:259-62.
18. Sommer SS. Cassady JD. Sobell J L. Botlema CD. A novel method for detecting point mutations or polymor phism s and its applica tion to population screening for carriers of phenylketonuria. Mayo Clin Proc 1989:64: 136 1-72.
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