CAJ | 학술논문

试验建立了鉴别猪伪狂犬病病毒（PRV）强弱毒高效纳米PCR检测方法，并对相关条件进行优化，组装了试剂盒。根据PRV保守序列设计3对引物分别扩增PRV基因组的gB（431 bp）、gE（316 bp）和gG（202 bp）3个基因，用于区分PRV强毒与基因缺失毒株，优化反应条件后建立了纳米PCR检测PRV强弱毒的方法并组装试剂盒，对试剂盒进行了特异性、敏感性、批内批间可重复性及保存期评估试验，并对临床样品进行检测。特异性试验表明，此试剂盒对于PRV能够扩增出431（gB）、316（gE）和202 bp（gG）的目的片段，对于PRV-Bartha-K61能够扩增出431和202 bp的目的片段，对于猪捷申病毒、非洲猪瘟病毒、猪圆环病毒2型、猪繁殖与呼吸综合征病毒、猪细小病毒及大肠杆菌等DNA或cDNA均未扩增出条带。敏感性试验表明，此试剂盒的方法比常规PCR方法敏感100～1000倍，最低核酸拷贝数检出量可以达到101 copy/μL数量级。试剂盒的批内、批间检测结果无明显差异，稳定性良好。-20℃至少可保存12个月。在对中国黑龙江、吉林等7个省市的临床送检的117份样品进行检测，结果显示，PRV强毒阳性率为51%，阴性率为49%，未发现有弱毒感染。鉴别PRV强弱毒纳米PCR试剂盒的研制，对PRV感染的早期检测、野毒株和疫苗株的鉴别、疾病控制等都有重要意义，而且也可以用于其他动物PRV感染的检测和早期诊断。
시험건립료감별저위광견병병독（PRV）강약독고효납미PCR검측방법，병대상관조건진행우화，조장료시제합。근거PRV보수서렬설계3대인물분별확증PRV기인조적gB（431 bp）、gE（316 bp）화gG（202 bp）3개기인，용우구분PRV강독여기인결실독주，우화반응조건후건립료납미PCR검측PRV강약독적방법병조장시제합，대시제합진행료특이성、민감성、비내비간가중복성급보존기평고시험，병대림상양품진행검측。특이성시험표명，차시제합대우PRV능구확증출431（gB）、316（gE）화202 bp（gG）적목적편단，대우PRV-Bartha-K61능구확증출431화202 bp적목적편단，대우저첩신병독、비주저온병독、저원배병독2형、저번식여호흡종합정병독、저세소병독급대장간균등DNA혹cDNA균미확증출조대。민감성시험표명，차시제합적방법비상규PCR방법민감100～1000배，최저핵산고패수검출량가이체도101 copy/μL수량급。시제합적비내、비간검측결과무명현차이，은정성량호。-20℃지소가보존12개월。재대중국흑룡강、길림등7개성시적림상송검적117빈양품진행검측，결과현시，PRV강독양성솔위51%，음성솔위49%，미발현유약독감염。감별PRV강약독납미PCR시제합적연제，대PRV감염적조기검측、야독주화역묘주적감별、질병공제등도유중요의의，이차야가이용우기타동물PRV감염적검측화조기진단。
In this study, a Nanoparticle-assisted Polymerase Chain Reaction(nanoPCR) kit was developed to detect and differentiate wild-type and gene-deleted pseudorabies strains ( PRV ) . Three primers here were designed from conserved regions of PRV with amplicon of gB (431 bp) gE (316 bp) gG (202 bp) and used for differentiate wild-type and gene-deleted PRV, and a nanoPCR kit was then developed with the specificity, sensitivity, repeatability and retention period tested after this method was optimized, then it was tested by clinical samples. DNA or cDNA of PRV, PRV-Bartha-K61, porcine teschovirus (PTV), African swine fever virus (ASFV), porcine circovirus type 2(PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), and Escherichia Coli(E.coli) were detected by this assay, results showed that the amplicon with size of 431 bp, 316 bp and 202 bp were amplified from PRV, 431 bp and 202 bp were amplified in the PRV-Bartha-K61. The sensitivity of the assay for this kit was 100~1 000 fold higher than conventional PCR. The intra- and inter-repeatability test showed no significant difference, and no significant change was found after being conserved at-20 ℃ for 12 months. Total of the 117 clinical samples collected from 7 provinces of China showed that 51%(60 of 117) were positive for wild-type PRV, and 49%(57 of 117) were negative. In conclusion, the nanoPCR kit should be a useful method for early and rapid detection of PRV and meanwhile differentiation of swine vaccinated with PRV gE-deleted vaccine from swine infected with wild virus, also of other kind of animals infected with PRV.