Reverse transcription-enzymatic recombinase amplification coupled with CRISPR-Cas12a for rapid detection and differentiation of PEDV wild-type strains and attenuated vaccine strains.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute watery diarrhea and vomiting in unweaned piglets, and is associated with high mortality, thus causing severe economic losses in the pig industry. Currently, although attenuated vaccines are commonly used in commercial pig farms in China, they do not completely protect against all mutated wild-type strains. Existing nucleic acid assays have high sensitivity and specificity, but the complexity of the assay process and expensive instrumentation hinder disease detection. Here, reverse transcription-enzymatic recombinase amplification (RT-ERA) was combined with the CRISPR-Cas12a system to develop a rapid diagnostic method to distinguish PEDV wild-type strains from attenuated vaccine strains. The protocol used crRNA and RT-ERA amplification primers against open reading frame 3 (ORF3), followed by Cas12a/crRNA complex detection of predefined target sequences at 37 °C for 30 min, thus producing results visible to the naked eye under LED blue light. The assay is highly sensitive and specific, detecting as few as two copies of the target gene per test and showing no cross-reactivity with other porcine pathogens. Overall, this integrated RT-ERA pre-amplification and Cas12a/crRNA cleavage assay is a practical tool for reliable and rapid detection of PEDV for diagnostic differentiation.

Simultaneous differentiation and diagnosis of goose parvovirus and astrovirus in clinical samples with duplex SYBR Green I real-time PCR.

Two pairs of primers were designed to bind conserved genomic regions of goose parvovirus (GPV) and goose astrovirus (GAstV) to establish a simple, sensitive, and highly specific duplex quantitative PCR (qPCR) method to simultaneously detect the two viruses. The duplex qPCR can distinguish GPV (melting point: 82.1 °C) and GAstV (melting point: 79.8 °C) by the peaks of their individual melting curves. Mixed testing with other waterfowl viruses produced no nonspecific peaks. The established standard curves showed good linear relationships (R2 > 0.997) and the limits of detection (LOD) for GPV and GAstV were 5.74 × 101 and 6.58 × 101 copies/µL, respectively. Both intra- and inter-assay coefficients of variation were <2%, indicating that the method has good repeatability. Twenty tissue samples from diseased geese were examined with the duplex qPCR assay and conventional PCR. Duplex qPCR showed positive rates of 25% for GPV and 45% for GAstV, and the positive rate for GPV and GAstV coinfection was 15%, slightly higher than the results for conventional PCR. These results indicated that this duplex qPCR method is highly sensitive, specific, and reproducible, and is suitable for epidemiological studies to effectively control the transmission of GPV and GAstV.

A TaqMan-based quantitative real-time PCR assay for identification of the goose circovirus.

Goose circovirus (GoCV) is a potential immunosuppressive virus that poses a great hazard to the goose industry and has been shown to be widely distributed throughout China. We have established a fast, sensitive and highly specific TaqMan real-time quantitative PCR detection method for this virus. Specific primers and probes were designed against the conserved regions of the genomic GoCV Rep gene. The results showed that the assay was highly specific and sensitive for GoCV and did not cross-react with other non-targeted waterfowl viruses. The established method will be helpful for epidemiological detection and may be effective in the prevention and control of the disease.

Epidemiology and Evolution of Emerging Porcine Circovirus-like Viruses in Pigs with Hemorrhagic Dysentery and Diarrhea Symptoms in Central China from 2018 to 2021.

Porcine circovirus-like virus (PCLV) is a type of circular Rep-encoding single-stranded DNA virus and may be associated with the development of diarrheal symptoms in pigs. In this study, we retrospectively analyzed three years of past cases in Anhui, China, and reported a case of hemorrhagic enteritis and death in a pregnant sow possibly caused by PCLV. In addition, we analyzed the evolutionary characteristics of PCLV and found that mutation, recombination and selective pressure all played an important role in the evolution of PCLV. We identified N15D and T17S as well as L56T, T58R, K59Q, M62R, L75I and R190K mutations in two different branches, and we noted recombination events in the Rep of a group of Chinese strains. Analysis of selection pressure revealed that PCLV gained more positive selection, indicating that the virus is in a continuous evolutionary state. The PR2 plot, ENC-plot and neutrality analysis showed a greater role of natural selection than that of mutational pressure in the formation of codon usage patterns. This study is the first to identify PCLV in sows with hemorrhagic dysentery and death, and it provides new epidemiological information on PCLV infection in pigs in China.