MALDI-TOF nucleic acid mass spectrometry for simultaneously detection of fourteen porcine viruses and its application.

BACKGROUND: Mixed infections of multiple viruses significantly contribute to the prevalence of swine diseases, adversely affecting global livestock production and the economy. However, effectively monitoring multiple viruses and detecting mixed infection samples remains challenging. This study describes a method that combines single-base extension PCR with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to detect important porcine viruses. RESULTS: Our approach accurately and simultaneously identified 14 porcine viruses, including porcine circovirus types 1-3, porcine bocaviruses groups 1-3, African swine fever virus, pseudorabies virus, porcine parvovirus, torque teno sus virus, swine influenza virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, and foot-and-mouth disease virus. The low limit of detection for multiplex identification ranges from 13.54 to 1.59 copies/µL. Inter- and intra-assay stability was found to be ≥98.3 %. In a comprehensive analysis of 114 samples, the assay exhibited overall agreement with qPCR results of 97.9 %. CONCLUSIONS: The developed MALDI-TOF NAMS assay exhibits high sensitivity, specificity, and reliability in detecting and distinguishing a wide spectrum of porcine viruses in complex matrix samples. This underscores its potential as an efficient diagnostic tool for porcine-derived virus surveillance and swine disease control.

A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3.

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide. We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap and developed a multiplex crystal digital PCR (cdPCR) method after optimizing the primer concentration, probe concentration, and annealing temperature. The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.

Verification of Bacteroidales 16S rRNA markers as a complementary tool for detecting swine fecal pollution in the Yangtze Delta.

To correctly assess and properly manage the public health risks associated with exposure to contaminated water, it is necessary to identify the source of fecal pollution in a watershed. In this study, we evaluated the efficacy of our two previously developed real time-quantitative PCR (qPCR) assays for the detection of swine-associated Bacteroidales genetic markers (gene 1-38, gene 3-53) in the Yangtze Delta watershed of southeastern China. The results indicated that the gene 1-38 and 3-53 markers exhibited high accuracy (92.5%, 91.7% conditional probability, respectively) in detecting Bacteroidales spp. in water samples. According to binary logistic regression (BLR), these two swine-associated markers were well correlated (P < 0.05) with fecal indicators (Escherichia coli and Enterococci spp.) and zoonotic pathogens (E. coli O157: H7, Salmonella spp. and Campylobacter spp.) in water samples. In contrast, concentrations of conventional fecal indicator bacteria (FIB) were not correlated with zoonotic pathogens, suggesting that they are noneffective at detecting fecal pollution events. Collectively, the results obtained in this study demonstrated that a swine-targeted qPCR assay based on two Bacteroidales genes markers (gene 1-38, gene 3-53) could be a useful tool in determining the swine-associated impacts of fecal contamination in a watershed.

Validation and application of quantitative PCR assays using host-specific Bacteroidales genetic markers for swine fecal pollution tracking.

Genome fragment enrichment (GFE) method was applied to identify host-specific bacterial genetic markers that differ among different fecal metagenomes. To enrich for swine-specific DNA fragments, swine fecal DNA composite (n = 34) was challenged against a DNA composite consisting of cow, human, goat, sheep, chicken, duck and goose fecal DNA extracts (n = 83). Bioinformatic analyses of 384 non-redundant swine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode metabolism-associated, cellular processes and information storage and processing. After challenged against fecal DNA extracted from different animal sources, four sequences from the clone libraries targeting two Bacteroidales- (genes 1-38 and 3-53), a Clostridia- (gene 2-109) as well as a Bacilli-like sequence (gene 2-95), respectively, showed high specificity to swine feces based on PCR analysis. Host-specificity and host-sensitivity analysis confirmed that oligonucleotide primers and probes capable of annealing to select Bacteroidales-like sequences (1-38 and 3-53) exhibited high specificity (>90%) in quantitative PCR assays with 71 fecal DNAs from non-target animal sources. The two assays also demonstrated broad distributions of corresponding genetic markers (>94% positive) among 72 swine feces. After evaluation with environmental water samples from different areas, swine-targeted assays based on two Bacteroidales-like GFE sequences appear to be suitable quantitative tracing tools for swine fecal pollution.