Shi Kaichuang, Hu Xin, Yin Yanwen, Shi Yuwen, Pan Yi, Long Feng, Feng Shuping, Li Zongqiang
School of Basic Medical Sciences, Youjiang Medical University for Nationalities, Baise, China.
College of Animal Science and Technology, Guangxi University, Nanning, China.
Front Vet Sci. 2024 Dec 12;11:1462880. doi: 10.3389/fvets.2024.1462880. eCollection 2024.
Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), and classical swine fever virus (CSFV) are currently prevalent worldwide and cause similar neurological symptoms in infected pigs. It is very important to establish a detection method that can rapidly and accurately detect and differentiate these three viruses. Targeting the PHEV N gene, PRV gB gene, and CSFV 5' untranslated region (5'UTR), three pairs of specific primers and probes were designed, and a triplex crystal digital reverse transcription-PCR (cdRT-PCR) was developed to detect PHEV, PRV, and CSFV. The results indicated that this assay had high sensitivity, and the limitation of detection (LODs) for PHEV, PRV, and CSFV were 4.812, 4.047, and 5.243 copies/reaction, respectively, which was about 50 times higher than that of multiplex real-time quantitative RT-PCR (RT-qPCR). This assay showed good specificity, without cross-reaction with other important swine pathogens, i.e., FMDV, PRRSV, PEDV, SIV, TGEV, PoRV, and PCV2. This assay had high repeatability, with intra-assay coefficients of variation (CVs) of 0.73-1.87%, and inter-assay CVs of 0.57-2.95%. The developed assay was used to test 1,367 clinical tissue samples from Guangxi province in China, and the positive rates of PHEV, PRV, and CSFV were 3.44% (47/1,367), 1.24% (17/1,367), and 1.90% (26/1,367), respectively, with a coincidence rate of 98.98% and a Kappa value of 0.94 to the reference multiplex RT-qPCR. The established triplex cdRT-PCR was a highly rapid, sensitive, and accurate assay to detect and differentiate PHEV, PRV, and CSFV.
猪血凝性脑脊髓炎病毒（PHEV）、猪伪狂犬病病毒（PRV）和经典猪瘟病毒（CSFV）目前在全球范围内流行，可在感染猪中引起相似的神经症状。建立一种能够快速、准确地检测和区分这三种病毒的检测方法非常重要。针对PHEV N基因、PRV gB基因和CSFV 5'非翻译区（5'UTR），设计了三对特异性引物和探针，并开发了一种三重晶体数字逆转录PCR（cdRT-PCR）来检测PHEV、PRV和CSFV。结果表明，该检测方法具有高灵敏度，PHEV、PRV和CSFV的检测限（LOD）分别为4.812、4.047和5.243拷贝/反应，比多重实时定量RT-PCR（RT-qPCR）高约50倍。该检测方法具有良好的特异性，与其他重要的猪病原体，即口蹄疫病毒（FMDV）、猪繁殖与呼吸综合征病毒（PRRSV）、猪流行性腹泻病毒（PEDV）、猪流感病毒（SIV）、猪传染性胃肠炎病毒（TGEV）、猪轮状病毒（PoRV）和猪圆环病毒2型（PCV2）无交叉反应。该检测方法具有高重复性，批内变异系数（CV）为0.73-1.87%，批间CV为0.57-2.95%。所开发的检测方法用于检测来自中国广西的1367份临床组织样本，PHEV、PRV和CSFV的阳性率分别为3.44%（47/1367）、1.24%（17/1367）和1.90%（26/1367），与参考多重RT-qPCR的符合率为98.98%，Kappa值为0.94。所建立的三重cdRT-PCR是一种检测和区分PHEV、PRV和CSFV的高度快速、灵敏和准确的检测方法。
