A multiplex RT-PCR assay for rapid and simultaneous detection of four RNA viruses in swine

Abstract

A B S T R A C TA multiplex reverse transcription polymerase chain rection (mRT-PCR) was developed for simultaneous detection of four RNA viruses in swine. The conserved target sequences directed to classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis coronavirus (TGEV) were selected based on alignments of genomic sequences and then specific primers were designed. The mRT-PCR assay was developed and evaluated for its specificity and sensitivity. The expected product from the single viral template was amplified by mRT-PCR and no spurious PCR amplification occurred from the genomic RNA or DNA of other pathogens. For single virus or different combinations of two viruses the detection limit of mRT-PCR was consistent with a single RT-PCR wtith 1 × 10 3 copies. For different combinations of the three viruses or four viruses, sensitivity of PEDV detection partially decreased. All of positive clinical specimens by the mRT-PCR were identically confirmed using Taqman RT-qPCR. Therefore, the mRT-PCR is a useful tool for epidemiological studies and laboratory diagnosis of single virus and/or mixed infections in swine.

Development of a TaqMan-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses

“…The LOD of our multiplex real-time PCR detection method can reach as low as 1 × 10 2 copies/μL for each pathogen, while that of the singleplex conventional RT-PCR is generally around 1 × 10 3 copies/μL to 1 × 10 4 copies/μL [ 51 ]. Likewise, multiplex conventional RT-PCR shows no noticeable advantage in terms of sensitivity [ 34 , 52 ]. Hui et al .…”

“…developed a multiplex RT-PCR detection for CSFV, porcine reproductive and respiratory syndrome virus (PRRSV), PEDV, and TGEV. The limit of detection of this method was 1 × 10 3 copies/μL [ 52 ]. On the other hand, although multiplex real-time PCR combines high sensitivity and high detection efficiency, the design of qualified multiplex real-time PCR assays, especially of quadruplex quantitative real-time PCR assays, is challenging [ 48 , 49 , 53 ].…”

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

“…Our results indicated that samples with a minimum of 10 4 to 10 6 viral copies, depending on the sample type, can be successfully sequenced to accurately identify strains after about 6 hours of sequencing. Although DRS is not as sensitive as PCR for use as a diagnostic tool identifying viral presence [77,78], it can be used for further investigation of the strain causing an outbreak, either directly from high viral load samples or following amplification of virus in cell culture. Additionally, a very strong correlation was observed between the number of viral reads generated through sequencing and the starting number of viral copies, indicating sequencing reads can be predicted by viral copies in a sample and vice versa, which has been confirmed by other studies as well [28].…”

Recent Advances in the Diagnosis of Classical Swine Fever and Future Perspectives

“…It is quite useful and convenient for quick and accurate detection of different pathogens in mixed infections, which is common in swine production systems. Multiplex RT-PCR assays for rapid detection and genotyping of CSFVs [ 36 , 37 ], simultaneous detection, and differentiation of common swine viruses [ 38 , 39 , 40 , 41 ] have been developed. Additionally, multiplex combined high-throughput molecular diagnostic platform, user-friendly electronic microarray, magnetoelastic sensor, and microfluidic detection systems were developed as potential alternatives for detection and surveillance of CSFV infection [ 42 , 43 , 44 , 45 , 46 ].…”