Use of dual priming oligonucleotide system-based multiplex RT-PCR assay to detect five diarrhea viruses in pig herds in South China

Abstract

In this study, a specific and simple method based on the dual priming oligonucleotide (DPO) system was developed to simultaneously detect transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus A (PRV-A), porcine delta coronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), associated with the major enteric RNA viruses in pigs. The DPO system-based multiplex RT-PCR method simplified the primer design and did not require optimization of the annealing temperature. Specificity analysis revealed that the method could specifically detect TGEV, PEDV, PRV-A, PDCoV, and SADS-CoV without any cross-amplification of other circulating swine viruses. The limit of detection of the method was as low as 103–104 copies/μL plasmid of each virus. The method also had good repeatability, and obvious results were seen in three repeat experiments with an interval of 45 days. This optimized multiplex RT-PCR method was used to evaluate 181 clinical swine samples that were collected from four provinces of China between September 2016 and August 2018. The results showed that the positive detection rates of PEDV, PDCoV, SADS-CoV, PRV-A, and TGEV were 30.94% (56/181), 17.67% (32/181), 11.6% (21/181), 9.39% (17/181), and 0.55% (1/181), respectively. Mixed infection of two or more viruses was also common. The DPO system-based multiplex RT-PCR could be a useful tool for detecting enteric virus infections. This method has the advantages of easy operation, low cost, high detection efficiency, and short running time for early diagnosis in clinical cases.

Current State of Molecular and Serological Methods for Detection of Porcine Epidemic Diarrhea Virus

“…In particular, the primer pairs should be not complementary to each other. In order to block nonspecific priming, a novel dual-priming oligonucleotide (DPO) system was recently used in multiplex RT-PCR assays for the specific detection of PEDV and other swine viruses, including TGEV, PRV-A, PDCoV and swine acute diarrhea syndrome coronavirus (SADS-CoV) [ 90 , 91 ]. The DPO primers have a special structure which is different from that of conventional primers.…”

“…Moreover, using the DPO primers, optimization of the annealing temperature is not required since annealing temperatures already differ for the two primer fragments as a function of their structure. This simplifies primer design [ 90 , 91 ]. Because coronaviruses evolve rapidly, the selection of the most suitable pair of primers is a matter of critical importance.…”

PEAC: An Ultrasensitive and Cost-Effective MRD Detection System in Non-small Cell Lung Cancer Using Plasma Specimen

“…To accurately detect low-level ctDNA and MRD, we thoroughly investigated available ctDNA detection technologies, such as widely used Amplification Refractory Mutation System (ARMS), digital PCR (dPCR)/digital droplet PCR (ddPCR) (16,17), Next-generation sequencing (NGS) technology (18,19), and several blocker sequence-based PCR enrichment methods (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). ARMS-PCR is clinically affordable, but it can only detect one mutation per assay and has a limit of detection ranging between ∼0.1 and 1% using 4 ml plasma (39,40).…”

“…Although optimized to achieve high sensitivity, these NGS technologies require high sequencing depth (>10,000X) and is costly during periodical MRD testing. In consideration of both detection limits and accumulated MRD testing costs, we reviewed other simple, sensitive, and cost-effective technologies, which include peptide nucleic acid (PNA)-based method ( 23 , 29 , 34 , 36 ), locked nucleic acid (LNA)-based method ( 21 , 22 , 25 , 26 , 28 , 31 , 32 ), dual priming oligonucleotide (DPO)-based method ( 20 , 24 , 27 , 30 , 33 , 35 , 37 ), and blocker displacement amplification (BDA) ( 38 ). Except for BDA, the mentioned approaches used modified blocker sequence with outperformed affinity to bind with the complementary sequence other than the sequence with a certain mutation.…”

Establishment of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of swine acute diarrhea syndrome coronavirus (SADS-CoV)

“…At present, the main methods for diagnosing SADS-CoV are reverse transcription PCR (RT-PCR) [ 23 ], quantitative real-time PCR [ 24 – 26 ], reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) [ 27 , 28 ], virus isolation culture, immunofluorescence (IF) and immunohistochemistry (IHC), with the gold standard and most common method for the diagnosis of SADS-CoV being nucleic acid testing technology. But some nucleic acid testing methods are time-consuming and complex and require expensive instruments and professional technicians, so it is very important to establish an economical, simple and efficient detection method.…”