Dataset Information

Comprehensive multiplex one-step real-time TaqMan qRT-PCR assays for detection and quantification of hemorrhagic fever viruses.

ABSTRACT:

Background

Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection.

Results

Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens.

Conclusions

Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive, stable and easy to serve as a useful tool for rapid detection of HFVs.

SUBMITTER: Pang Z

PROVIDER: S-EPMC3994070 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Publications

Comprehensive multiplex one-step real-time TaqMan qRT-PCR assays for detection and quantification of hemorrhagic fever viruses.

Pang Zheng Z Li Aqian A Li Jiandong J Qu Jing J He Chengcheng C Zhang Shuo S Li Chuan C Zhang Quanfu Q Liang Mifang M Li Dexin D

PloS one 20140421 4

<h4>Background</h4>Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of ...[more]

PMID: 24752452

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Project description:BackgroundAfrican swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused great economic losses to the swine industry in China. Since coinfections of ASFV, CSFV and APPV occur in certain pig herds, it is necessary to accurately and differentially detect these pathogens in field-collected samples. In this study, a one-step multiplex real-time quantitative reverse transcription-polymerase chain reaction (multiplex qRT-PCR) was developed for the simultaneous and differential detection of ASFV, CSFV and APPV.ResultsThe one-step multiplex qRT-PCR presented here was able to simultaneously detect ASFV, CSFV and APPV but could not amplify other viruses, including porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), porcine parvovirus (PPV), porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PRoV), porcine deltacoronavirus (PDCoV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV-1), BVDV-2, etc. The limit of detection (LOD) of the assay was 2.52 × 101 copies/μL for ASFV, CSFV and APPV. A repeatability test using standard recombinant plasmids showed that the intra- and interassay coefficients of variation (CVs) were less than 2%. An assay of 509 clinical samples collected in Guangxi Province, southern China, from October 2018 to December 2020 showed that the positive rates of ASFV, CSFV and APPV were 45.58, 12.57 and 3.54%, respectively, while the coinfection rates of ASFV and CSFV, ASFV and APPV, CSFV and APPV were 4.91, 1.38, 0.98%, respectively. Phylogenetic analysis based on the nucleotide sequences of the partial ASFV p72 gene showed that all ASFV strains from Guangxi Province belonged to genotypes I and II.ConclusionA one-step multiplex qRT-PCR with high specificity, sensitivity and repeatability was successfully developed for the simultaneous and differential detection of ASFV, CSFV and APPV.

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Dataset Information

Comprehensive multiplex one-step real-time TaqMan qRT-PCR assays for detection and quantification of hemorrhagic fever viruses.

Background

Results

Conclusions

Publications

Comprehensive multiplex one-step real-time TaqMan qRT-PCR assays for detection and quantification of hemorrhagic fever viruses.

Similar Datasets

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