Development of a GeXP-multiplex PCR assay for the simultaneous detection and differentiation of six cattle viruses

Abstract

Foot-and-mouth disease virus (FMDV), Bluetongue virus (BTV), Vesicular stomatitis Virus (VSV), Bovine viral diarrheal (BVDV), Bovine rotavirus (BRV), and Bovine herpesvirus 1 (IBRV) are common cattle infectious viruses that cause a great economic loss every year in many parts of the world. A rapid and high-throughput GenomeLab Gene Expression Profiler (GeXP) analyzer-based multiplex PCR assay was developed for the simultaneous detection and differentiation of these six cattle viruses. Six pairs of chimeric primers consisting of both the gene-specific primer and a universal primer were designed and used for amplification. Then capillary electrophoresis was used to separate the fluorescent labeled PCR products according to the amplicons size. The specificity of GeXP-multiplex PCR assay was examined with samples of the single template and mixed template of six viruses. The sensitivity was evaluated using the GeXP-multiplex PCR assay on serial 10-fold dilutions of ssRNAs obtained via in vitro transcription. To further evaluate the reliability, 305 clinical samples were tested by the GeXP-multiplex PCR assay. The results showed that the corresponding virus specific fragments of genes were amplified. The detection limit of the GeXP-multiplex PCR assay was 100 copies/μL in a mixed sample of ssRNAs containing target genes of six different cattle viruses, whereas the detection limit for the Gexp-mono PCR assay for a single target gene was 10 copies/μL. In detection of viruses in 305 clinical samples, the results of GeXP were consistent with simplex real-time PCR. Analysis of positive samples by sequencing demonstrated that the GeXP-multiplex PCR assay had no false positive samples of nonspecific amplification. In conclusion, this GeXP-multiplex PCR assay is a high throughput, specific, sensitive, rapid and simple method for the detection and differentiation of six cattle viruses. It is an effective tool that can be applied for the rapid differential diagnosis of clinical samples and for epidemiological investigation.

Meta-Analysis of qPCR for Bovine Respiratory Disease Based on MIQE Guidelines

“…All studies included primer/probe sequence data or clearly referenced cited studies. All articles included both analytical sensitivity and specificity and only one study included diagnostic sensitivity and specificity ( Andres-Lasheras et al, 2020 ); ten studies reported on PCR optimization ( Horwood and Mahony, 2011 ; Thonur et al, 2012 ; Nefedchenko et al, 2016 ; Fan et al, 2017 ; Zhang et al, 2017 ; Loy et al, 2018 ; Thanthrige-Don et al, 2018 ; Liu et al, 2019 ; Andres-Lasheras et al, 2020 ; Xie et al, 2021 ) and three studies reported the use of quantitative PCR methods; qPCR ( Andres-Lasheras et al, 2020 ), Multiplex qRT-PCR ( Goto et al, 2020 ) and RT-qPCR ( Xie et al, 2021 ). Quantitative values were not reported in any of the studies included in this analysis ( Table 3 ).…”

Simultaneous Detection of Bovine Rotavirus, Bovine Parvovirus, and Bovine Viral Diarrhea Virus Using a Gold Nanoparticle-Assisted PCR Assay With a Dual-Priming Oligonucleotide System

“…They all primarily infect newborn calves and cause diarrhea (Rigo-Adrover et al, 2019). Mixed infections cause severe diarrhea that leads to increased mortality (Ruiz et al, 2015;Fan et al, 2017) and make clinical diagnosis difficult. Therefore, it is imperative to develop a diagnostic method that can simultaneously detect multiple pathogens resulting in enhanced epidemic surveillance.…”

Simultaneous Differentiation of the N1 to N9 Neuraminidase Subtypes of Avian Influenza Virus by a GeXP Analyzer-Based Multiplex Reverse Transcription PCR Assay

“…GenomeLab Gene Expression Profiler (GeXP) technology represents a novel platform for high-throughput nucleic acid detection based on multiplex RT-PCR assays and an analysis of amplicons size using capillary electrophoresis, a method that is capable of differentially assessing the expression profile of up to 30 genes in one tube by converting amplification with multiple primers to amplification with a pair of universal primers. The GeXP analyzer has been widely adopted in veterinary diagnostics and medical examinations, and displays high sensitivity and specificity (Hu et al, 2012; Li et al, 2012; Zeng et al, 2015; Zhang et al, 2015; Fan et al, 2017). Currently, a method for the rapid and high-throughput diagnosis of nine NA subtypes is urgently needed.…”