Development and use of a droplet digital PCR (ddPCR) assay to achieve sensitive and fast atypical porcine pestivirus detection.

Atypical porcine pestivirus (APPV) is a recently discovered RNA virus, which mainly caused congenital tremor in piglets. Droplet digital PCR (ddPCR) is an absolute quantitative method that does not rely on the standard curve but has high sensitivity and accuracy. The present study aimed to develop a ddPCR detection assay for APPV. Furthermore, we evaluated the limit of detection, sensitivity, specificity and reproducibility of the ddPCR and real-time quantitative PCR (qPCR) and tested 135 clinical samples to calculate the detection rate of the two methods. The results showed that both methods had a strong linear relationship and quantitative correlation. The ddPCR assay had a minimum detection limit of 0.15 copies/µL for APPV, with a sensitivity 100 times that of qPCR. We tested clinical samples and found that the APPV ddPCR had a 27.4% positive detection rate, noticeably higher than that of the qPCR (14.8%). Additionally, the APPV ddPCR method had excellent repeatability and specificity. In brief, our study provided a novel, feasible and sensitive diagnostic technique to identify and monitor APPV.

Evaluation on the efficacy and immunogenicity of recombinant DNA plasmids expressing S gene from porcine epidemic diarrhea virus and VP7 gene from porcine rotavirus.

Porcine rotavirus (PoRV) and porcine epidemic diarrhea virus (PEDV) usually co-infect pigs in modern large-scale piggery, which both can cause severe diarrhea in newborn piglets and lead to significant economic losses to the pig industry. The VP7 protein is the main coat protein of PoRV, and the S protein is the main structural protein of PEDV, which are capable of inducing neutralizing antibodies in vivo. In this study, a DNA vaccine pPI-2.EGFP.VP7.S co-expressing VP7 protein of PoRV and S protein of PEDV was constructed. Six 8-week-old mice were immunized with the recombinant plasmid pPI-2.EGFP.VP7.S. The high humoral immune responses (virus specific antibody) and cellular immune responses (IFN-γ, IL-4, and spleen lymphocyte proliferation) were evaluated. The immune effect through intramuscular injection increased with plasmid dose when compared with subcutaneous injection. The immune-enhancing effect of IFN-α adjuvant was excellent compared with pig spleen transfer factor and IL-12 adjuvant. These results demonstrated that pPI-2.EGFP.VP7.S possess the immunological functions of the VP7 proteins of PoRV and S proteins of PEDV, indicating that pPI-2.EGFP.VP7.S is a candidate vaccine for porcine rotaviral infection (PoR) and porcine epidemic diarrhea (PED).

Rapid and sensitive detection of Bordetella bronchiseptica by loop-mediated isothermal amplification (LAMP)

Bordetella bronchiseptica causes acute and chronic respiratory infections in diverse animal species and occasionally in humans. In this study, we described the establishment of a simple, sensitive and cost-efficient loop-mediated isothermal amplification (LAMP) assay for the detection of B. bronchiseptica. A set of primers towards a 235 bp region within the flagellum gene of B. bronchiseptica was designed with online software.. The specificity of the LAMP assay was examined by using 6 porcine pathogens and 100 nasal swabs collected from healthy pigs and suspect infected pigs. The results indicated that positive reactions were confirmed for all B. bronchiseptica and no cross-reactivity was observed from other non-B. bronchiseptica. In sensitivity evaluations, the technique successfully detected a serial dilutions of extracted B. bronchiseptica DNA with a detection limit of 9 copies, which was 10 times more sensitive than that of PCR. Compared with conventional PCR, the higher sensitivity of LAMP method and no need for the complex instrumentation make this LAMP assay a promising alternative for the diagnosis of B. bronchiseptica in rural areas and developing countries where there lacks of complex laboratory services.

Rapid and sensitive detection of Bordetella bronchiseptica by loop-mediated isothermal amplification (LAMP)

Bordetella bronchiseptica causes acute and chronic respiratory infections in diverse animal species and occasionally in humans. In this study, we described the establishment of a simple, sensitive and cost-efficient loop-mediated isothermal amplification (LAMP) assay for the detection of B. bronchiseptica. A set of primers towards a 235 bp region within the flagellum gene of B. bronchiseptica was designed with online software.. The specificity of the LAMP assay was examined by using 6 porcine pathogens and 100 nasal swabs collected from healthy pigs and suspect infected pigs. The results indicated that positive reactions were confirmed for all B. bronchiseptica and no cross-reactivity was observed from other non-B. bronchiseptica. In sensitivity evaluations, the technique successfully detected a serial dilutions of extracted B. bronchiseptica DNA with a detection limit of 9 copies, which was 10 times more sensitive than that of PCR. Compared with conventional PCR, the higher sensitivity of LAMP method and no need for the complex instrumentation make this LAMP assay a promising alternative for the diagnosis of B. bronchiseptica in rural areas and developing countries where there lacks of complex laboratory services. (AU)

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene

This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP) for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63ºC for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection limit of the conventional PCR was 10² CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene

This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP) for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63ºC for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection limit of the conventional PCR was 10² CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.(AU)