摘要
建立了定量检测猪圆环病毒DNA的竞争PCR方法.应用PCR扩增PCV-2 ORF2上490 bp片段P3P4,克隆到pMD 18-T载体获得目标模板.构建含692 bp的c-P3P4 DNA片段的竞争模板,其携带与目标DNA和靶DNA相同的引物结合区.以定量的竞争模板同一系列稀释的竞争模板进行竞争PCR扩增,以产物的荧光强度(IOD)绘制标准曲线,结果当反应体系中起始模板的量为1.0×107 cop/μL,循环次数小于或等于30时,原始模板数以指数增长.由此确定目标模板和竞争模板的最适工作浓度并用该方法检测感染细胞内的PCV-2基因组的拷贝数和临床样品中的PCV-2 DNA含量,结果发现临床样品中的PCV-2 DNA含量达到一定程度(肺中1.0145×108 copies/mL,血中0.8×107 copies/mL,淋巴中9.698×108 copies/mL),猪表现临床症状,而病毒在感染细胞内的复制在80 h基因的拷贝数达到最高,这为今后的研究奠定了基础.
Competitive quantitative PCR(cPCR) assay was developed for monitoring and detecting porcine circovirus (PCV-2) DNA. Using PCR assay, the cPCR was based on competitive co-amplification of a 490 bp region of the PCV-20RF2 gene, with a known concentration of competitor DNA, which produced a 692 bp fragment. The cPCR was validated by quantification of a known amount of competitor PCV DNA with one series of dilutions of target DNA. Using the regression equation, we protracted the standard curve and affirmed the quantity of the competitive template. We used this technique to determine PCV genome copy numbers in infected cells. Furthermore, we measured PCV DNA loads in clinical sample and found PCV2 DNA content in the piglet with PMS is higher than 1.0145 × 10~8 copies/ml in lung, 0.8 × 10~7 copies/mL in blood, and 9.698 × 10~8 copies/mL in lymph. Our research might provide some hints to the study of the future work.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2004年第5期347-351,共5页
Chinese Journal of Preventive Veterinary Medicine
