Multiplex digital PCR: a superior technique to qPCR for the simultaneous detection of duck Tembusu virus, duck circovirus, and new duck reovirus.

Duck Tembusu virus (DTMUV), duck circovirus (DuCV), and new duck reovirus (NDRV) have seriously hindered the development of the poultry industry in China. To detect the three pathogens simultaneously, a multiplex digital PCR (dPCR) was developed and compared with multiplex qPCR in this study. The multiplex dPCR was able to specifically detect DTMUV, DuCV, and NDRV but not amplify Muscovy duck reovirus (MDRV), Muscovy duck parvovirus (MDPV), goose parvovirus (GPV), H4 avian influenza virus (H4 AIV), H6 avian influenza virus (H6 AIV), and Newcastle disease virus (NDV). The standard curves showed excellent linearity in multiplex dPCR and qPCR and were positively correlated. The sensitivity results showed that the lowest detection limit of multiplex dPCR was 1.3 copies/µL, which was 10 times higher than that of multiplex qPCR. The reproducibility results showed that the intra- and interassay coefficients of variation were 0.06-1.94%. A total of 173 clinical samples were tested to assess the usefulness of the method; the positive detection rates for DTMUV, DuCV, and NDRV were 18.5, 29.5, and 14.5%, respectively, which were approximately 4% higher than those of multiplex qPCR, and the kappa values for the clinical detection results of multiplex dPCR and qPCR were 0.85, 0.89, and 0.86, indicating that the two methods were in excellent agreement.

Development and application of a multiplex qPCR assay for the detection of duck circovirus, duck Tembusu virus, Muscovy duck reovirus, and new duck reovirus.

A multiplex qPCR assay was developed to simultaneously detect duck circovirus (DuCV), duck Tembusu virus (DTMUV), Muscovy duck reovirus (MDRV), and novel duck reovirus (NDRV), but it did not amplify other viruses, including duck virus enteritis (DVE), infectious bursal disease virus (IBDV), avian reovirus (ARV), H5 avian influenza virus (H5 AIV), H7 avian influenza virus (H7 AIV), H9 avian influenza virus (H9 AIV), Newcastle disease virus (NDV), and Muscovy duck parvovirus (MDPV), and the detection limit for DuCV, DTMUV, MDRV, and NDRV was 1.51 × 101 copies/µL. The intra- and interassay coefficients of variation were less than 1.54% in the repeatability test with standard plasmid concentrations of 1.51 × 107, 1.51 × 105, and 1.51 × 103 copies/µL. The developed multiple qPCR assay was used to examine 404 clinical samples to verify its practicability. The positivity rates for DuCV, DTMUV, MDRV, and NDRV were 26.0%, 9.9%, 4.0%, and 4.7%, respectively, and the mixed infection rates for DuCV + DTMUV, DuCV + MDRV, DuCV + NDRV, MDRV + NDRV, DTMUV + MDRV, and DTMUV + NDRV were 2.7%, 1.2%, 1.2%, 1.0%, 0.5%, and 0.7%, respectively.

A Quadruplex qRT-PCR for Differential Detection of Four Porcine Enteric Coronaviruses.

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) are four identified porcine enteric coronaviruses. Pigs infected with these viruses show similar manifestations of diarrhea, vomiting, and dehydration. Here, a quadruplex real-time quantitative PCR (qRT-PCR) assay was established for the differential detection of PEDV, TGEV, PDCoV, and SADS-CoV from swine fecal samples. The assay showed extreme specificity, high sensitivity, and excellent reproducibility, with the limit of detection (LOD) of 121 copies/µL (final reaction concentration of 12.1 copies/µL) for each virus. The 3236 clinical fecal samples from Guangxi province in China collected between October 2020 and October 2022 were evaluated by the quadruplex qRT-PCR, and the positive rates of PEDV, TGEV, PDCoV, and SADS-CoV were 18.26% (591/3236), 0.46% (15/3236), 13.16% (426/3236), and 0.15% (5/3236), respectively. The samples were also evaluated by the multiplex qRT-PCR reported previously by other scientists, and the compliance rate between the two methods was more than 99%. This illustrated that the developed quadruplex qRT-PCR assay can provide an accurate method for the differential detection of four porcine enteric coronaviruses.

The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus.

African swine fever virus (ASFV) causes African swine fever (ASF), a devastating hemorrhagic disease of domestic pigs and wild boars. Currently, the MGF505R, EP402R (CD2v) and I177L gene-deleted ASFV strains were confirmed to be the ideal vaccine candidate strains. To develop an assay for differentiating the wild-type and gene-deleted ASFV strains, four pairs of specific primers and TaqMan probes targeting the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes were designed. A multiplex real-time qPCR assay for the differential detection of the wild-type and gene-deleted ASFV strains was developed after optimizing the reaction conditions, including the annealing temperature, primer concentration and probe concentration. The results showed that the multiplex real-time qPCR assay can specifically test the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes with a limit of detection (LOD) of 32.1 copies/µL for the B646L (p72) gene, and 3.21 copies/µL for the I177L, MGF505-2R and EP402R (CD2v) genes. However, the assay cannot test for the classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine circovirus type 2 (PCV2), PCV3 and pseudorabies virus (PRV). The assay demonstrated good repeatability and reproducibility with coefficients of variation (CV) less than 1.56% for both the intra- and inter-assay. The assay was used to test 4239 clinical samples, and the results showed that 12.60% (534/4239) samples were positive for ASFV, of which 10 samples lacked the EP402R gene, 6 samples lacked the MGF505-2R gene and 14 samples lacked the EP402R and MGF505-2R genes. The results indicated that the multiplex real-time qPCR developed in this study can provide a rapid, sensitive and specific diagnostic tool for the differential detection of the ASFV B646L, I177L, MGF505-2R and EP402R genes.

Metabolomics Characterize the Differential Metabolic Markers between Bama Xiang Pig and Debao Pig to Identify Pork.

The Bama Xiang pig (BM) is a unique pig species in Guangxi Province, China. Compared to other breeds of domestic pig, such as the Debao pig (DB), it is smaller in size, better in meat quality, resistant to rough feeding and strong in stress resistance. These unique advantages of Bama Xiang pigs make them of great edible value and scientific research value. However, the differences in muscle metabolites between Bama Xiang pigs (BM) and Debao pigs (DB) are largely unexplored. Here, we identified 214 differential metabolites between these two pig breeds by LC-MS. Forty-one such metabolites are enriched into metabolic pathways, and these metabolites correspond to 11 metabolic pathways with significant differences. In Bama pigs, the abundance of various metabolites such as creatine, citric acid, L-valine and hypoxanthine is significantly higher than in Debao pigs, while the abundance of other metabolites, such as carnosine, is significantly lower. Among these, we propose six differential metabolites: L-proline, citric acid, ribose 1-phosphate, L-valine, creatine, and L-arginine, as well as four potential differential metabolites (without the KEGG pathway), alanyl-histidine, inosine 2'-phosphate, oleoylcarnitine, and histidinyl hydroxyproline, as features for evaluating the meat quality of Bama pigs and for differentiating pork from Bama pigs and Debao pigs. This study provides a proof-of-concept example of distinguishing pork from different pig breeds at the metabolite level and sheds light on elucidating the biological processes underlying meat quality differences. Our pork metabolites data are also of great value to the genomics breeding community in meat quality improvement.