Apple bull’s eye rot is caused by pathogenic Neofabraea species including N. malicorticis, N. perennans and N. alba. Fruits carrying this fungal quarantine disease are prohibited from entering China. The host plants ...Apple bull’s eye rot is caused by pathogenic Neofabraea species including N. malicorticis, N. perennans and N. alba. Fruits carrying this fungal quarantine disease are prohibited from entering China. The host plants for the pathogens include several Rosaceae fruits including apple and pear. Disease symptoms and pathogen morphology are often insufficient to determine the identity of the pathogen, particularly at the species level. In the current study, we analyzed the inter-species sequence variations in the β-tublin gene, and designed specific primers to allow PCR amplification of 554 bp fragments from pathogenic Neofabraea species. The PCR products were recovered and sequenced, and Blast search was conducted using the DNA sequences in the Genbank database. The results indicated precise PCR amplification of the target sequences from the host pathogen, which allowed unambiguous identification of the species.展开更多
Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to des...Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false<span style="font-family:;" "=""> </span><span style="font-family:Verdana;">negatives, and benefitting early diagnosis.</span>展开更多
文摘Apple bull’s eye rot is caused by pathogenic Neofabraea species including N. malicorticis, N. perennans and N. alba. Fruits carrying this fungal quarantine disease are prohibited from entering China. The host plants for the pathogens include several Rosaceae fruits including apple and pear. Disease symptoms and pathogen morphology are often insufficient to determine the identity of the pathogen, particularly at the species level. In the current study, we analyzed the inter-species sequence variations in the β-tublin gene, and designed specific primers to allow PCR amplification of 554 bp fragments from pathogenic Neofabraea species. The PCR products were recovered and sequenced, and Blast search was conducted using the DNA sequences in the Genbank database. The results indicated precise PCR amplification of the target sequences from the host pathogen, which allowed unambiguous identification of the species.
文摘Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false<span style="font-family:;" "=""> </span><span style="font-family:Verdana;">negatives, and benefitting early diagnosis.</span>