Dataset Information

TaqMan-based real-time polymerase chain reaction assay for specific detection of bocavirus-1 in domestic cats.

ABSTRACT: Feline bocavirus-1 (FBoV-1) was first discovered in Hong Kong in 2012, and studies have indicated that the virus may cause feline hemorrhagic enteritis. Currently, there is a lack of an effective and quantitative method for FBoV-1 detection. In this study, a TaqMan-based quantitative real-time PCR (qPCR) for FBoV-1 detection was established. Primers and probes were designed to target the conserved region of the FBoV-1 NS1 gene. The sensitivity analysis indicated that the minimum detection limit was 4.57 × 101 copies/?L. The specificity test revealed no cross-reaction with seven other common feline viruses, including the same species-FBoV-2 and FBoV-3. The sensitivity of this method was 100 times higher than that of conventional PCR (cPCR). The established method showed good repeatability, with the intra-assay and inter-assay coefficients of variation of 0.18%-1.00% and 0.27%-0.45%, respectively. Furthermore, the analysis of feline feces revealed that the detection rate by qPCR was 7.0% (9/128), whereas that by cPCR was 4.7% (6/128). In conclusion, the established qPCR assay can quantitatively detect FBoV-1 with a high sensitivity, high specificity, and good reproducibility, making it a promising technique for the clinical detection of and basic and epidemiological research on FBoV-1.

SUBMITTER: Wang Y

PROVIDER: S-EPMC7414301 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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TaqMan-based real-time polymerase chain reaction assay for specific detection of bocavirus-1 in domestic cats.

Wang Yong Y Sun Jianfei J Guo Xu X Zhang Da D Cui Yongqiu Y Li Wei W Liu Guangqing G Li Yongdong Y Jiang Shudong S

Molecular and cellular probes 20200808

Feline bocavirus-1 (FBoV-1) was first discovered in Hong Kong in 2012, and studies have indicated that the virus may cause feline hemorrhagic enteritis. Currently, there is a lack of an effective and quantitative method for FBoV-1 detection. In this study, a TaqMan-based quantitative real-time PCR (qPCR) for FBoV-1 detection was established. Primers and probes were designed to target the conserved region of the FBoV-1 NS1 gene. The sensitivity analysis indicated that the minimum detection limit ...[more]

PMID: 32777447

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Project description:The definitive diagnosis of lyssavirus infection (including rabies) in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR) based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR) method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan) RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR) and a second reaction using an intercalating dye (SYBR Green) to detect other lyssavirus species (pan-lyssa RT-qPCR). The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135) including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5%) and saliva (54%) samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco). This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for lyssavirus surveillance.

Project description:Background and objectivesThe variable response of hepatitis C virus (HCV) genotypes towards anti-viral treatment requires prior information on the genotype status before planning a therapeutic strategy. Although assays for typing or subtyping of HCV are available, however, a fast and reliable assay system is still needed. The present study was planned to develop a single-step multiplex quantitative real time polymerase chain reaction (qPCR) assay to determine HCV genotypes in patients' sera.MethodsThe conserved sequences from 5' UTR, core and NS5b regions of HCV genome were used to design primers and hydrolysis probes labeled with fluorophores. Starting with the standardization of singleplex (qPCR) for each individual HCV-genotype, the experimental conditions were finally optimized for the development of multiplex assay. The sensitivity and specificity were assessed both for singleplex and multiplex assays. Using the template concentration of 102 copies per microliter, the value of quantification cycle (Cq) and the limit of detection (LOD) were also compared for both singleplex and multiplex assays. Similarly, the merit of multiplex assay was also compared with sequence analysis and restriction fragment length polymorphism (RFLP) techniques used for HCV genotyping. In order to find the application of multiplex qPCR assay, it was used for genotyping in a panel of 98 sera positive for HCV RNA after screening a total number of 239 patients with various liver diseases.ResultsThe results demonstrated the presence of genotype 1 in 26 of 98 (26.53%) sera, genotype 3 in 65 (66.32%) and genotype 4 in 2 (2.04%) sera samples, respectively. One sample showed mixed infection of genotype 1 and 3. Five samples could not show the presence of any genotype. Genotypes 2, 5 and 6 could not be detected in these sera samples. The analysis of sera by singleplex and RFLP indicated the results of multiplex to be comparable with singleplex and with clear merit of multiplex over RFLP. In addition, the results of multiplex assay were also found to be comparable with those from sequence analysis. The sensitivity, specificity, Cq values and LOD values were compared and found to be closely associated both for singleplex and multiplex assays.ConclusionThe multiplex qPCR assay was found to be a fast, specific and sensitive method that can be used as a technique of choice for HCV genotyping in all routine laboratories.

Dataset Information

TaqMan-based real-time polymerase chain reaction assay for specific detection of bocavirus-1 in domestic cats.

Publications

TaqMan-based real-time polymerase chain reaction assay for specific detection of bocavirus-1 in domestic cats.

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