Specific Detection of Enterovirus 71 Directly From Clinical

8/2/2019 Specific Detection of Enterovirus 71 Directly From Clinical

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Specific Detection Of

Enterovirus 71 Directly From

Clinical Specimens

Using Real-time RT-PCR

Hybridization Probe AssayEng Lee Tan, Vincent Tak Kwong Chow, Gamini Kumarasinghe,

Raymond Tze Pin Lin,

Ian M. MacKay , Theo P. Sloots , Chit Laa Poh


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Hand, Foot And Mouth Disease

HFMD

A mild, contagious viral infection common in

young children

It presents as a vesicular eruption in the mouth.

HFMD usually affects infants and children,

and is quite common.

The usual incubation period is 3

7 days. It typically occurs in small epidemics


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Pathophysiology

Infection generally occurs via the fecal-oral route orvia contact with skin lesions and oral secretions.

The virus spreads to regional lymph nodes within 24hours.

Viraemia rapidly follows

Followed by invasion of the skin and mucousmembranes.

The incubation period is 4 to 7 days

However, there may be a prodromal period of 3 to 4days.

Lesions in the mouth heal within 1 week, and lesionson the hands and feet may last for up to 10 days.


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Symptoms

Fever

Sore throat

Headache

Malaise

Vomiting Fatigue

Referred ear pain

Irritability in infants and toddlers

Loss of appetite Diarrhea

A red rash, without itching but sometimes with blistering,on the palms, soles and sometimes the buttocks


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Causative Agent

Coxsackievirus A type 16 (CV A16) is the

etiologic agent involved in most cases of HFMD

The illness is also associated with coxsackievirus

A5, A7, A9, A10, B2, and B5 strains.

Enterovirus 71(EV-71) has also caused outbreaks

of HFMD with associated neurologic involvement

in the western Pacific region.


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Picornaviruses

Pico means small

They are small positive strand RNA viruses

Non-enveloped

Spherical

About 30 nm in diameter Composed of a protein shell surrounding the

naked RNA genome.

The capsid consists of a densely-packed icosahedral

arrangement of 60 protomers. Each consisting of 4 polypeptides, VP1, VP2, VP3 and VP4.

VP4 is located on the internal side of the capsid.


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There are nine genera within the

Picornaviridae.

Five of these infect humans:

Enteroviruses

Rhinoviruses Hepatoviruses

Parechoviruses

Kobuviruses


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GENOME

ssRNA(+) genome of 7.2-8.5 kb

The structural polypeptides.The nonstructural proteins associated withreplication.


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Enterovirus 71

It was first discovered and isolated from thestool of an infant who was suffering fromencephalitis in California in 1969

Australia was the first country Clinically, HFMD caused by EV71 is

indistinguishable from that caused by

Coxsackievirus A16 (CA16) Diagnosis depends largely on virus isolation

and serotyping.


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Virus Strains

A neurovirulent EV71 strain, 7423/MS/87 and a CA16strain (CA16-G-10)

Two Singapore strains, the fatal 5865/SIN/00009 strain(designated as Strain 41) and the non-fatal

5666/SIN/002209 strain (designated as Strain 10) wereisolated from patients during the outbreak in October 2000and cultivated in tissue cultures.

A total of 55 clinical specimens obtained from pediatricpatients suffering from HFMD during the period from 2000

to 2003 in Singapore were also included in this study. Other enterovirus isolates analyzed included CA24,

Coxsackieviruses B1 (CB1), CB2, CB3, CB4, CB5 andEchoviruses 4,6,7,18,19,24,30.


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Clinical Samples

The clinical specimens included CSF, stool,

rectal swabs, blood, throat swabs and urine

specimens.

The CSF and urine specimens were processed

immediately upon receiving them.

A 10% stool suspension was made.

Blood Samples

Addition 0.5 g ofstool (0.5 ml for fluid

stools) to 5 ml ofphosphate buffered

saline at pH 7.4.

The suspension wasthen centrifuged at13,000g for 10 min

and filtered.

The filtrate was thensubsequently

processed.

Stand in a vertical position forabout 1520 min.

Centrifuging for 10min at 13,000g

The serum thenaspirated and

transferred to a clean1.5 ml sterile

eppendorf tube.


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RNA Extraction

Viral RNA extraction was carried out using

QIAampw Viral RNA Mini Kit (Qiagen,

Valencia, USA)

The specimens were first lysed with a buffer

The RNA released bound to the membrane.

The bound RNA is washed twice with wash buffers to

remove any contaminating proteins and lipids.

The RNA was then eluted with an elution buffer


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Design of EV71 primers and probes

The VP1 nucleotide sequences of 6 EV71 strainsand 2 CA16 strains from the GenBank wereanalyzed.

Using BLAST together with DNASTAR program,a highly conserved VP1 region of EV71 strainsthat differed from CA16 was defined.

For the real-time RTPCR hybridization probeanalysis, two primers (designated as EvVP1F and

EvVP1R) and two hybridization probes(designated as EvVP1-FL and EvVP1-LC) weredesigned.


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RT-PCR


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Real Time PCR


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Real Time PCR

As the name suggests, real time PCR is a technique used to monitorthe progress of a PCR reaction in real time.

Real Time PCR is based on the detection of the fluorescenceproduced by a reporter molecule which increases, as the reactionproceeds.

These fluorescent reporter molecules include dyes that bind to thedouble-stranded DNA (i.e. SYBR Green ) or sequence specificprobes (i.e. Molecular Beacons or TaqMan Probes).

Real time PCR facilitates the monitoring of the reaction as itprogresses.

One can start with minimal amounts of nucleic acid and quantify theend product accurately.

Real time PCRassays are now easy to perform, have high sensitivity,more specificity, and provide scope for automation.


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Real Time PCR Using LightCycler

The LightCycler 480 real-time PCR system is

a multiwell platebased instrument that

provides integrated applications for detecting

and characterizing genetic variation.

The glass capillaries of rapid cycling make

ideal fluorescence cuvettes that are arranged in

a carousel and rotated past stationary optics.


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The signal from these adjacent hybridization probes dependsdirectly on hybridization, not on exonuclease activity and probehydrolysis.

Each probe is covalently labeled with only one dye, so they areinherently simpler to synthesize than double-labeled probes.

One probe is labeled on the 3'-end. The other probe is labeled on the 5'-end and its 3'-end blocked to

prevent extension.

When both probes are hybridized, fluorescence resonance energytransfer occurs.

Maximum fluorescence occurs with a one base separation betweenprobes.

The fluorescence signal is even more specific than single probesystems because hybridization of 4 oligonucleotides (2 primers and2 probes) is required.


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RT-PCR in glass capillaries using the LightCyclerinstrument.

The enzyme mix contains a mixture of reversetranscriptase and Faststart Taq Polymerase thatallows reverse transcription of RNA template andsubsequent cDNA amplification.

Each 10 ul reaction contained

cDNA was first synthesized from the RNA byreverse transcription

Melting curve analysis was also carried out afterthe PCR reaction.

1.0 ml of RNA,

5 mM MgCl2,

0.5 mM of the forward primer,

0.3 mM of reverse primer,0.2 mM of each hybridization

probe,

2.0 ml RNA hybridization

probe amplification reaction

solution,

0.2 ml enzyme mix and water.


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Different EV71 strains

Numbers 1

3 represent the amplifications of strains 41, 10 and 7423/MS/87, respectively. Noamplification was observed for other enterovirus serotypes (Number 4).


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Hybridization probe melting curve analysis of the amplicons generated from three different

EV71 viral isolates. The melting curves of strains 41, 10 and 7423/MS/87 were represented byNumbers 13, respectively.


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Discussion

The real-time RT-PCR assay was able to detect the presence ofEV71 in 46 out of the 55 clinical specimens.

The cell culture method and a conventional semi-nested RT-PCRamplification of the VP1 region of EV71 were

For the cell culture method, none of the 20 clinical specimens tested

positive for EV71. However, amplification of the VP1 region was observed for the

same 20 clinical specimens

No EV71 RNA was detected in nine clinical specimens by the real-time RT-PCR hybridization probe assay.

To determine whether these specimens contained EV71,conventional seminested RT-PCR was conducted with these nineclinical specimens and no amplification of the VP1 region wasobserved


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Conclusion

Real-time RT-PCR assay could be completed within 2 h upon

The real-time PCR assay was also shown to be more sensitive than the cell culturemethod as the assay was able to detect EV71 at far lower concentrations.

The primers and probes were designed based on the VP1 region of the EV71genome as it is known to possess a high degree of antigenic and genetic diversitythat may be useful in distinguishing between enterovirus serotypes.

In the melting curve analysis, since the two hybridization probes are nothydrolyzed during amplification, the Light- Cycler is able to monitor thefluorescence changes during the denaturation of the probes from their templates.

This allows fluorescence melting curves to be generated and provide significantinformation about the sequence that the probes bind to.

The results from this study showed that the real-time RT-PCR hybridization probe-based approach can be used diagnostically to discriminate single nucleotide

polymorphisms in the DNA templates. Determination of the Tms from melting curve analysis of EV71 viral amplicons

will be helpful in elucidating phylogenetic relationships of the strains isolated in anoutbreak or from different outbreaks.


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Thank You

Documents

Specific Detection of Enterovirus 71 Directly From Clinical