1345 - JOURNAL OF CLINICAL MICROBIOLOGY, May 1993,p....

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Unformatted text preview: JOURNAL OF CLINICAL MICROBIOLOGY, May 1993,p. 1345-1349 Vol. 31,No. 5 0095-1137/93/051345-05$02.00/0 Copyright 1993, American Society for Microbiology Nested Polymerase Chain Reaction forHigh-Sensitivity DetectionofEnteroviral RNA inBiologicalSamples GIOVANNI MARIA SEVERINI,1 LUISA MESTRONI,1'2 ARTURO FALASCHI,1 FULVIO CAMERINI,2 A N D MAURO GIACCAl* InternationalCentreforGeneticEngineeringandBiotechnology, ARE4 SciencePark; Padriciano 99,34012 Trieste, and Departmentof Cardiology,Ospedale Maggiore and University, 34100 Trieste,2 Italy Received 6November 1992/Accepted2February 1993 A methodbased on nestedpolymerasechain reaction was developed forthedetectionofenteroviral genomes inbiologicalsamples.By takingadvantageoftheconserved5'noncodingregionoftheenteroviral RNA, two setsof primers were utilized,enablingthedetectioneitherofa broad range ofenterovimses or ofgroup B coxsackievirusesonly. The sensitivityofthemethod isclosetothedetectionofsinglemoleculesofviralRNA inas muchas 1 m g oftissuesample.A preliminarystudyshowedtheusefulnessofthistechniquefortheanalysis ofendomyocardialbiopsysamplesfrom patientswithidiopathicdilatedcardiomyopathyand myocarditis. Inthepicornavirusfamily,severalmembersoftheentero- virus(EV) genus (includingpolio-,echo-, and coxsackievi- ruses)are importantpathogensforhumans(18).Diagnosisof EV infection usually requires propagation ofvirus in cell cultures followed by neutralization typing with antiserum pools; thisprocedure, however, islaborious and time-con- sumingandoftenappears tohaveinadequatesensitivity(22), especially in diseases such as idiopathic myocarditis and idiopathicdilatedcardiomyopathy, forwhich epidemiologi- calandexperimentaldatasuggesta coxsackievirusinfection butviralisolationusuallyfails(20). Taking advantage of the availability of the complete nucleotidesequences ofover 30human picornavirusstrains (reviewed by Stanway [28]), we have developed a method based on nestedpolymerase chainreaction(PCR)withtwo differentsetsofoligonucleotides abletorecognizeeitherall the EVs or specifically group B coxsackieviruses. This method does not require an internalhybridizationstep and hasa sensitivityclosetothedetectionofa singlemoleculeof viralRNA present in1 m g oftissuesample. Nested PCR amplificationforEV-specificand coxsackievi- m s group B-specificdetection.Seven differentprimers were synthesized for reverse transcription-PCR amplification of EV genomes (seeFig.1A forlocalizationand 1B forprimer sequences). All the primers are from the 5' noncoding region, which contains sequence homology boxes highly conserved among differentEVs, some ofwhich have been usedbydifferentauthorsforhybridization(2,10,23,24)and PCR studies(5,7,9,13,21,22,31)....
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1345 - JOURNAL OF CLINICAL MICROBIOLOGY, May 1993,p....

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