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Objective To develop and evaluate a single -tube multiplex RT -real time PCR assay for the simultaneous detection of rotavirus,astrovirus and hepatitis A virus.Methods Gen -bank sequences of rotavirus,astrovirus and hepatitis A virus were included as reference sequences.Primers and probes were designed based on the reference sequences.A multiplex real -time RT -PCR assay was developed,and the reproducibility,specificity and sensitivity of the assay were evaluated.Fecal samples from 1 28 patients with viral diarrhea were detected and verified by gene sequencing.Results There was high reproducibility and specificity of the single -tube multiplex real -time RT -PCR assay for detecting rotavirus,astrovirus and hepatitis A virus.Detection limits of 1 01 copies were achieved in tests of rotavirus,1 02 copies for astrovirus and hepatitis A virus in one reaction.3.1 3% and 1 7.97% of 1 28 clinical specimens were detected positive for astrovirus RNA and rotavirus RNA respectively.And the positive samples were verified by gene sequencing.Conclusion Rotavirus,astrovirus and hepatitis A virus can be detected and identified by the single -tube multiplex RT -real time PCR assay with high specificity and sensitivity.The assay developed in this study can be applied to the clinical diagnosis and epidemiological investigation.
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<p><b>OBJECTIVE</b>The purpose of this study was to develop RT- PCR assay for Rapidly detect and distinguish between Norovirus genogroup I and genogroup II with a pair of primers.</p><p><b>METHODS</b>A pairs of primers specific to capsid prote in ORF2 gene of G I and G II Norovirus were dsigned according to the published complete genome sequence, with which the RNA of Norovirus was extracted and RT-PCR amplification. The sensitivity, specificity of the RT- PCR assay was estimated and apply it to the detection of Norovirus in clinical specimens.</p><p><b>RESULTS</b>The results showed that the assay possessed high specificity for Norovirus detection and without any evident cross-reaction with other viruses, including rotavirus, enteric adenovirus and hepatitis A virus. The detection limit of RT-PCR assay for Norovirus G I and G II were up to 100 pg/ml and 10 pg/ml respectively.</p><p><b>CONCLUSION</b>The RT- PCR assay provide rapid and sensitive detection of Norovirus G I and G II and should prove to be useful for Norovirus diagnosis in the outbreaks of acute gastroenteritis.</p>
Subject(s)
Humans , Caliciviridae Infections , Diagnosis , Virology , DNA Primers , Genetics , Gastroenteritis , Diagnosis , Virology , Norovirus , Classification , Genetics , Reverse Transcriptase Polymerase Chain Reaction , MethodsABSTRACT
To study the molecular characteristics of Noroviruses causing outbreaks of acute gastroenteritis in Huzhou. During April 2008 and February 2009, fecal specimens of patients collected from 2 outbreaks of acute gastroenteritis were tested for Norovirus by real-time RT-PCR. Partial sequence of RNA dependent RNA polymerase(RdRp) of the positive samples were amplified by RT-PCR, the PCR products were then purified, sequenced and phylogenetic analysis was conducted. Both genogroup II (GII) and genogroup I (GI) noroviruses were detected in 2 outbreaks. Phylogenetic analysis revealed that two of the GI norovirus strains isolated from 2008 belonged to genotype GI/2 and one of the GI Norovirus strain isolated from 2009 belonged to genotype GI/3. The other GIIú norovirus strains isolated from 2009 had high nucleotide identity with GIIb genotype that had been reported frequently in European countries during 2000 and 2001 and in Asian countries recently. These results suggested that the epidemic strains of norovirus isolated in Huzhou had a high degree of genetic diversity and prevalent genotypes at different times were also different. To our knowledge this is the first report of detecting GIIb variant in outbreaks of acute gastroenteritis in China.
Subject(s)
Humans , Acute Disease , Caliciviridae Infections , Epidemiology , Virology , China , Epidemiology , Disease Outbreaks , Feces , Virology , Gastroenteritis , Epidemiology , Virology , Genotype , Norovirus , Classification , Genetics , Phylogeny , RNA, Viral , Genetics , Sequence Homology, Nucleic AcidABSTRACT
<p><b>OBJECTIVE</b>To study the molecular characteristics of Noroviruses causing outbreaks of acute gastroenteritis in Huzhou.</p><p><b>METHODS</b>From 2008 to 2010, total 119 fecal specimens collected from outbreaks of acute gastroenteritis were tested for Norovirus. Partial sequence of RNA dependent RNA polymerase (RdRp) of the positive samples were amplified by RT-PCR, then the PCR production were purified, sequenced and put into phylogenetic analysis.</p><p><b>RESULTS</b>50 of 119 specimens were positive for Norovirus by real-time RT-PCR. Out of those 50 Norovirus positive specimens, 9 were Norovirus Genogroup I (GI) positive, 35 were Norovirus Genogroup II (GII) positive, 6 was both Norovirus GI and GII positive. 12 PCR products for RdRp were selected for further studies on sequencing. Phylogenetic analysis revealed that the 5 GI norovirus isolates were belonged to genotype GI/2 and GI/3. Of the 7 GII norovirus isolates, 6 were belonged to genotype GII/4, 1 was belonged to genotype Glib.</p><p><b>CONCLUSION</b>Norovirus is a major cause of outbreaks of acute gastroenteritis in Huzhou and the epidemic strains of norovirus isolated from Huzhou had a high degree of genetic diversity.</p>
Subject(s)
Female , Humans , Male , Acute Disease , China , Epidemiology , Disease Outbreaks , Gastroenteritis , Epidemiology , Genetic Variation , Norovirus , Classification , Genetics , Phylogeny , RNA-Dependent RNA Polymerase , Genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
<p><b>OBJECTIVE</b>To detect HBV-LP and HBV DNA of the patients with hepatitis B, and to study the sensitive and specificity of HBV-LP detecting and its evaluate on for clinical application.</p><p><b>METHODS</b>The ELISA was used for HBV-LP detecting and RT-PCR for HBV DNA detecting.</p><p><b>RESULTS</b>The sensitive and specificity of HBV-LP and HBV DNA were 64.89%, 99.68%, 60.63% and 100% respectively (P > 0.05); +LR, -LR were 202.78, 60.63, 0.3522 and 0.3937, and there were significance between +LR of the detecting (P < 0.005).</p><p><b>CONCLUSION</b>The sensitive and specificity of HBV-LP and HBV DNA detecting are considerable, +LR of HBV-LP are higher comparing HBV DNA. The HBV-LP is better serology index for detecting replication of HBV DNA.</p>