Enhanced detection of African swine fever virus in samples with low viral load using digital PCR technology.

Detection of low viral load samples has long been a challenge for African swine fever (ASF) prevention and control. This study aimed to compare the detection efficacy of droplet digital PCR(ddPCR) and quantitative PCR(qPCR) for African swine fever virus (ASFV) at different viral loads, with a focus on assessing the accuracy of ddPCR in detecting low viral load samples. The results revealed that ddPCR had a detection limit of 1.97 (95% CI 1.48 - 4.12) copies/reaction and was 18.99 times more sensitive than qPCR (detection limit: 37.42, 95% CI 29.56 - 69.87 copies/reaction). In the quantification of high, medium, and low viral load samples, ddPCR showed superior stability with lower intra- (2.06% - 7.58%) and inter-assay (3.83% - 7.50%) coefficients of variation than those of qPCR (intra-assay: 8.08%-29.86%; inter-assay: 9.27%-34.58%). Bland-Altman analysis indicated acceptable consistency between ddPCR and qPCR for high and medium viral load samples; however, discrepancies were observed for low viral load samples, where two samples (2/24, 8.33%) exhibited deviations beyond the acceptable range (-46.18 copies/reaction). Moreover, ddPCR demonstrated better performance in detecting ASFV in clinical samples from asymptomatic pigs and environmental samples, with qPCR showing false negative rates of 7.69% (2/26) and 27.27% (12/44), respectively. McNemar analysis revealed significant differences between the two methods (P = 0.000) for samples with a viral load <100 copies/reaction. The results of this study demonstrate that ddPCR has better detection limits and adaptability than qPCR, allowing for a more accurate detection of ASFV in early-stage infections and low-concentration environmental samples. These findings highlight the potential of ddPCR in the prevention and control of ASF.

Overexpression of miR-101-2 in donor cells improves the early development of Holstein cow somatic cell nuclear transfer embryos.

Accumulating studies have suggested that microRNA play a part in regulating multiple cellular processes, such as cell proliferation, apoptosis, the cell cycle, and embryo development. This study explored the effects of miR-101-2 on donor cell physiological status and the development of Holstein cow somatic cell nuclear transfer (SCNT) embryos in vitro. Holstein cow bovine fetal fibroblasts (BFF) overexpressing miR-101-2 were used as donor cells to perform SCNT; then, cleavage rate, blastocyst rate, inner cell mass-to-trophectoderm ratio, and the expression of some development- and apoptosis-related genes in different groups were analyzed. The miR-101-2 suppressed the expression of inhibitor of growth protein 3 (ING3) at mRNA and protein levels, expedited cell proliferation, and decreased apoptosis in BFF, suggesting that ING3, a target gene of miR-101-2, is a potential player in this process. Moreover, by utilizing donor cells overexpressing miR-101-2, the development of bovine SCNT embryos in vitro was significantly enhanced; the apoptotic rate in SCNT blastocysts was reduced, and the inner cell mass-to-trophectoderm ratio and SOX2, POU5F1, and BCL2L1 expression significantly increased, whereas BAX and ING3 expression decreased. Collectively, these findings suggest that miR-101-2 promotes BFF proliferation and vitality, reduces their apoptosis, and improves the early development of SCNT embryos.

Simple and rapid detection of Salmonella serovar Enteritidis under field conditions by loop-mediated isothermal amplification.

AIM: The objective of this study is to develop a serovar-specific loop-mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions. METHODS: A set of six specific primers was designed with Salmonella Enteritidis DNA as the target. LAMP conditions were optimized by incubating the target DNA with the Bst DNA polymerase large fragment in a simple water bath. The sensitivity and specificity of LAMP was then compared with those of fluorescent quantitative real-time polymerase chain reaction (FQ-PCR). RESULTS: The results were as follows. (1) Serovar-specific Salmonella Enteritidis DNA was amplified at 65°C in as early as 20min in a water bath. (2) A colour change visible to the naked eye indicated a positive amplification reaction. (3) The detection limit of the LAMP assay was 4 copies µl(-1) ; thus, the sensitivity and specificity of this assay is similar to those of the FQ-PCR. CONCLUSIONS: LAMP is a high-throughput detection technique with high sensitivity, specificity, and simplicity; these factors make it suitable for specifically detecting Salmonella Enteritidis under field conditions and in laboratory settings. Thus, LAMP eliminates the need for complicated equipment and technical training in the detection of this specific serovar. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study involving the use of LAMP to detect Salmonella serovar-specific DNA sequences. It is also the first to report an ideal method of distinguishing between Salmonella Enteritidis and other Salmonella under field conditions.

Attenuated lapinized chinese strain of classical swine fever virus: complete nucleotide sequence and character of 3'-noncoding region.

The complete nucleotide sequence including precise 5'- and 3'-terminal non-coding regions (NCRs) of the attenuated lapinized Chinese strain (HCLV) of Classical Swine Fever Virus (CSFV) was determined from overlapping cDNA clones constructed by separated RT-PCR and rapid amplification of cDNA ends (RACE) methods. The genomic RNA of the HCLV strain consists of 12.310 nucleotides (nts) including 374 nts and 242nts in the 5'- and 3'-NCRs, respectively. It contains one large open reading frame (ORF) encoding a polyprotein of 3,898 amino acids with a calculated molecular weight of 437.6 kDa. There is one notable insertion of 12 continuous nts, CTTTTTTCTTTT in the 3'-NCR of HCLV genomic cDNA when compared with its parental virulent Shimen strain. Sequence alignment of partial 3'-NCR reveals two groups of CSFV vaccine strains carrying similar T-rich insertions at different positions in this region. Computer-predicted secondary structures suggest that T-rich insertion greatly change the structures and thus decrease the promoter functions of 3'-NCRs during the replications of these two groups of CSFV vaccine strains.