Development of a multiplex TaqMan qPCR assay for simultaneous detection and differentiation of four DNA and RNA viruses from clinical samples of sheep and goats

Xu, Xingang; Yang, Feng; Zhang, Qi; Xü, Ying; Huang, Jiana; Fu, Mingzhe; Zhang, Weiyang

doi:10.1016/j.jviromet.2019.01.015

Cited by 10 publications

(10 citation statements)

References 14 publications

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“…The multiplex assay showed that the probes were specific, and it was possible to discriminate the three phyla in a single tube. Similar studies reported that real-time PCR assays based on TaqMan hydrolysis probes were specific, sensitive, and rapid compared to conventional PCR [20,37,38]. It is important to note that our qPCR assay was based on the proportion of amplicons; therefore, it cannot directly measure the number of individual cells.…”

Section: Discussionmentioning

confidence: 74%

“…In addition, and of concern because the number of UC patients is rapidly increasing [36], it is more costly to monitor the clinical efficacy of FMT (i.e., analyzing the gut microbiota) of numerous people with NGS. A rapid, sensitive, and cost-effective method is required [20].…”

Section: Discussionmentioning

confidence: 99%

“…However, these methods are not able to simultaneously quantify different phyla in a single tube. TaqMan PCR is a powerful tool for the simultaneous detection and quantification of microbes in samples from different sources [18][19][20]. The relative composition of the three major phyla in the human gut, Bacteroidetes, Firmicutes, and Proteobacteria, has been proposed as a potential diagnostic tool for UC [9,21].…”

Section: Introductionmentioning

confidence: 99%

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In Situ Profiling of the Three Dominant Phyla Within the Human Gut Using TaqMan PCR for Pre-Hospital Diagnosis of Gut Dysbiosis

Tagele

Pham

et al. 2020

IJMS

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A microbial imbalance called dysbiosis leads to inflammatory bowel disease (IBD), which can include ulcerative colitis (UC). Fecal microbiota transplantation (FMT), a novel therapy, has recently been successful in treating gut dysbiosis in UC patients. For the FMT technique to be successful, the gut microbiota of both the healthy donors and UC patients must be characterized. For decades, next-generation sequencing (NGS) has been used to analyze gut microbiota. Despite the popularity of NGS, the cost and time constraints make it difficult to use in emergency services and activities related to the periodic monitoring of microbiota profile alterations. Hence, in this study, we developed a multiplex TaqMan qPCR assay (MTq-PCR) with novel probes to simultaneously determine the relative proportions of the three dominant microbial phyla in the human gut: Bacteroidetes, Firmicutes, and Proteobacteria. The relative proportions of the three phyla in fecal samples of either healthy volunteers or UC patients were similar when assessed NGS and the MTq-PCR. Thus, our MTq-PCR assay could be a practical microbiota profiling alternative for diagnosing and monitoring gut dysbiosis in UC patients during emergency situations, and it could have a role in screening stool from potential FMT donors.

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Section: Discussionmentioning

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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In Situ Profiling of the Three Dominant Phyla Within the Human Gut Using TaqMan PCR for Pre-Hospital Diagnosis of Gut Dysbiosis

Tagele

Pham

et al. 2020

IJMS

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“…A duplex (2-plex) TaqMan assay (Venkatesan et al 2014b) was reported for simultaneous detection and differentiation of SPV, GPV and ORFV but not LSDV or FMDV and the assay was evaluated without using any CS of cattle or IPC. A 4-plex TaqMan assay (Xu et al 2019) was reported for simultaneous detection and differentiation of SPV, ORFV and FMDV but not GPV or LSDV, and this assay also evaluated without using any CS of cattle or IPC. The newly developed multiplex assays described in this study were evaluated against all three differential viruses and their serotypes (CaPV, PaPV and FMDV) some of which were not included in the above studies.…”

Section: Discussionmentioning

confidence: 99%

“…Mixed infections with multiple viral pathogens are common and it can be devastating if any of the co-infecting pathogens is highly contagious, such as FMDV or CaPV. Mixed infections of FMDV or CaPV with other viruses in ruminants have been reported, including PaPV (Xu et al 2019;He et al 2017;Venkatesan et al 2014aVenkatesan et al & 2014bChu et al 2011), peste des petits ruminant virus (PPRV) (Kumar et al 2016), and blue tongue virus (BTV) (Malik et al 2011). Clinical diagnosis of mixed infections involving CaPV, PaPV, and FMDV can be challenging due to very similar clinical signs associated with these diseases.…”

Section: Introductionmentioning

confidence: 99%

Development of multiplex real-time PCR assays for differential detection of capripoxvirus, parapoxvirus, and foot-and-mouth disease virus

Das

Wang

Babiuk³

et al. 2020

Preprint

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This study reports the development of multiplex real-time PCR assays for differential detection of capripoxvirus (CaPV), parapoxvirus (PaPV), and foot-and-mouth disease virus (FMDV) in sheep, goats and cattle. Three multiplex assays were developed, a capripox (CaP) rule-out assay for simultaneous detection and differentiation of CaPV and PaPV, a FMD rule-out assay for simultaneous detection and differentiation of FMDV and PaPV, and a FMD/CaP rule-out assay for simultaneous detection and differentiation of CaPV, PaPV and FMDV. All multiplex assays included ?-actin gene ACTB as an internal positive control to monitor PCR inhibition and accuracy of nucleic acid extractions. The optimized assays were highly specific to the target viruses (CaPV, PaPV, and FMDV) with no cross-reactivity against other differential viruses. Using positive control plasmids as template, the limit of detection (LOD) of the multiplex assays were estimated as 2 (CaPV), 7 (PaPV), and 15 (FMDV) copies per assay. The amplification efficiency (AE) and correlation co-efficient (R2), estimated from the standard curves (Ct vs. log10 template dilution), were 94-106% and >0.99, respectively, for CaP and FMD rule-out assays, 96-116% (AE) and >0.98 (R2), respectively, for CaP/FMD rule-out assays and 91-102% and >0.99, respectively, for the corresponding singleplex assays. The diagnostic sensitivity (DSe) of the multiplex assays was assessed on 35 (CaPV), 36 (PaPV) and 39 (FMDV) clinical specimens collected from experimentally (CaPV and FMDV) and naturally (PaPV) infected animals, and all tested positive (DSe 100%) except two FMDV specimens that were tested negative (37/39; DSe 95%). The newly developed multiplex assays offer a valuable tool for differential detection of clinically indistinguishable CaPV, PaPV, and FMDV in suspected animals and animals with mixed infections.

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Recent advances in diagnostic approaches for orf virus

Pang

Long

2023

Appl Microbiol Biotechnol

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Orf virus (ORFV), the prototype species of the Parapoxvirus genus, is an important zoonotic virus, causing great economic losses in livestock production. At present, there are no effective drugs for orf treatment. Therefore, it is crucial to develop accurate and rapid diagnostic approaches for ORFV. Over decades, various diagnostic methods have been established, including conventional methods such as virus isolation and electron microscopy; serological methods such as virus neutralization test (VNT), immunohistochemistry (IHC) assay, immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA); and molecular methods such as polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and recombinase-aided amplification (RAA) assay. This review provides an overview of currently available diagnostic approaches for ORFV and discusses their advantages and limitations and future perspectives, which would be significantly helpful for ORFV early diagnosis and surveillance to prevent outbreak of orf. Key points• Orf virus emerged and reemerged in past years • Rapid and efficient diagnostic approaches are needed and critical for ORFV detection • Novel and sensitive diagnostic methods are required for ORFV detection Keywords Orf virus • Diagnostic approach • Conventional methods • Serological methods • Molecular methods

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Development of a multiplex TaqMan qPCR assay for simultaneous detection and differentiation of four DNA and RNA viruses from clinical samples of sheep and goats

Cited by 10 publications

References 14 publications

In Situ Profiling of the Three Dominant Phyla Within the Human Gut Using TaqMan PCR for Pre-Hospital Diagnosis of Gut Dysbiosis

In Situ Profiling of the Three Dominant Phyla Within the Human Gut Using TaqMan PCR for Pre-Hospital Diagnosis of Gut Dysbiosis

Development of multiplex real-time PCR assays for differential detection of capripoxvirus, parapoxvirus, and foot-and-mouth disease virus

Recent advances in diagnostic approaches for orf virus

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