支气管败血波氏杆菌SYBR GreenⅠ荧光定量PCR检测方法的建立
|
摘要
为建立支气管败血波氏杆菌SYBR GreenⅠ荧光定量PCR检测方法,本研究根据波氏菌属毒力基因DNT设计一对特异性引物,通过优化,确定最佳反应条件,建立了支气管败血波氏杆菌荧光定量PCR检测方法。并对其进行敏感性、特异性、重复性试验以及临床样品的检测。以支气管败血波氏杆菌标准质粒为模板,建立标准曲线结果显示:标准品浓度在1.13×10~8拷贝/μL～1.13×10~2拷贝/μL范围内具有良好的线性关系,相关数R~2=0.997;该方法的灵敏度可达1.13×10~1拷贝/μL。建立的荧光定量PCR方法与副猪嗜血杆菌、猪链球菌、金黄色葡萄球菌等12种细菌和伪狂犬病毒、猪瘟病毒等4种病毒无交叉反应,具有良好的特异性;批内变异系数(CV)均小于2%,批间变异系数均小于4%,具有良好的稳定性。对临床样品的检测结果显示该方法检测的阳性率为57.7%,与普通PCR的符合率为93.24%。本研究建立的方法可以用作支气管败血波氏杆菌感染的早期诊断、快速检测与定量分析。
To develop rapid and specific assay for Bordetella bronchiseptical detection, a SYBR GreenⅠbased real-time PCR method was established with a pair of primers designed according to the sequence of dermonecrotic toxingene(DNT) of B.bronchiseptical. Initially, a recombinant plasmid containing DNT gene was constructed and served as a standard template to generate the standard curve. The standard curve produced a linear relationship between Ct value and concentrations of the standard template at a range of 1.13 ×10~8 copies/μL to 1.13 ×10~2 copies/μL, R~2=0.997. Inaddition, the specificity and sensitivity of the method was analyzed through optimum reaction conditions. The specificity assay showed no amplification of 12 kinds of bacteria including Haemophilus parasuis, Streptococcus suis, Pasteurella multocidaetc and 4 kinds of viruses such as pseudorabies virus, classical swine fever virus etc, except B.bronchiseptical. and the sensitivity of this method was 1.13×10~1 copies/μL. The reproducibility test showed that the variation coefficients of intra-and inter-assay were less than 3% and 4%, respectively. For the detection of clinical samples, the consistent rate between conventional PCR and the real-time PCR(57.7% positive rate) 93.24%. Therefore, the established real-time PCR method is a rapid, specificand sensitive method for the detection B.bronchiseptical.
To develop rapid and specific assay for Bordetella bronchiseptical detection, a SYBR GreenⅠbased real-time PCR method was established with a pair of primers designed according to the sequence of dermonecrotic toxingene(DNT) of B.bronchiseptical. Initially, a recombinant plasmid containing DNT gene was constructed and served as a standard template to generate the standard curve. The standard curve produced a linear relationship between Ct value and concentrations of the standard template at a range of 1.13 ×10~8 copies/μL to 1.13 ×10~2 copies/μL, R~2=0.997. Inaddition, the specificity and sensitivity of the method was analyzed through optimum reaction conditions. The specificity assay showed no amplification of 12 kinds of bacteria including Haemophilus parasuis, Streptococcus suis, Pasteurella multocidaetc and 4 kinds of viruses such as pseudorabies virus, classical swine fever virus etc, except B.bronchiseptical. and the sensitivity of this method was 1.13×10~1 copies/μL. The reproducibility test showed that the variation coefficients of intra-and inter-assay were less than 3% and 4%, respectively. For the detection of clinical samples, the consistent rate between conventional PCR and the real-time PCR(57.7% positive rate) 93.24%. Therefore, the established real-time PCR method is a rapid, specificand sensitive method for the detection B.bronchiseptical.
引文
[1]裴洁,何华,赵战勤,等.支气管败血波氏杆菌的研究进展[J].畜牧兽医科技信息,2006,(02):4-6.
[2]李爽,元娜,张杏,等.猪支气管败血波氏杆菌的分离鉴定[J].中国兽医杂志,2016,52(04):51-53.
[3]覃娟娟.副猪嗜血杆菌和猪支气管败血波氏杆菌双重检测方法的建立及应用[D].成都:四川农业大学,2015.
[4]徐引弟,王治方,朱文豪,等.猪支气管败血波氏杆菌的分离鉴定和药敏试验[J].动物医学进展,2010,31(08):110-112.
[5]刘吉山,王艳,管宇,等.猪支气管败血波氏杆菌分离与鉴定[J].畜牧与饲料科学,2008,(01):29-31.
[6]裴洁.猪支气管败血波氏杆菌分离鉴定及疫苗菌株的生物学特性研究[D].武汉:华中农业大学,2006.
[7]Shen H G, Bender J S, Opriessnig T, et al. Identification of surface protective antigen(spa)types in Erysipelothrix reference strains and diagnostic samples by spa multiplex real-time and conventional assays[J]. J Appl Microbiol, 2010, 109(4):1227-1233.
[8]Hozbor D, Fouque F, Guiso N. Detection of Bordetella Bronchiseptica by the polymerase chain reaction[J]. Res Microbiol,1999, 150(5):333-341.
[9]胡慧,陈雅君,段志刚,等.大肠杆菌O157:H7特异基因的实时荧光定量PCR检测[J].食品科学,2011,32(12):278-282.
[10]肖海君,崔金生,翁立雪,等.支气管败血波氏杆菌PCR检测方法的建立和应用[J].山东农业大学学报(自然科学版),2009,40(01):70-74.
[11]邸海波,楚常欢,宋苍浩,等.支气管败血波氏杆菌、多杀性巴氏杆菌和副猪嗜血杆菌同时检测的PCR方法的建立[J].畜禽业,2010,(05):57-59.
[12]熊富强,王芳,恽时锋,等.兔支气管败血波氏杆菌双夹心ELISA检测方法的建立[J].江苏农业学报, 2010, 26(01):120-125.
[13]徐婷婷.荧光定量PCR技术应用综述[J].畜禽业,2010,(10):48-50.
[2]李爽,元娜,张杏,等.猪支气管败血波氏杆菌的分离鉴定[J].中国兽医杂志,2016,52(04):51-53.
[3]覃娟娟.副猪嗜血杆菌和猪支气管败血波氏杆菌双重检测方法的建立及应用[D].成都:四川农业大学,2015.
[4]徐引弟,王治方,朱文豪,等.猪支气管败血波氏杆菌的分离鉴定和药敏试验[J].动物医学进展,2010,31(08):110-112.
[5]刘吉山,王艳,管宇,等.猪支气管败血波氏杆菌分离与鉴定[J].畜牧与饲料科学,2008,(01):29-31.
[6]裴洁.猪支气管败血波氏杆菌分离鉴定及疫苗菌株的生物学特性研究[D].武汉:华中农业大学,2006.
[7]Shen H G, Bender J S, Opriessnig T, et al. Identification of surface protective antigen(spa)types in Erysipelothrix reference strains and diagnostic samples by spa multiplex real-time and conventional assays[J]. J Appl Microbiol, 2010, 109(4):1227-1233.
[8]Hozbor D, Fouque F, Guiso N. Detection of Bordetella Bronchiseptica by the polymerase chain reaction[J]. Res Microbiol,1999, 150(5):333-341.
[9]胡慧,陈雅君,段志刚,等.大肠杆菌O157:H7特异基因的实时荧光定量PCR检测[J].食品科学,2011,32(12):278-282.
[10]肖海君,崔金生,翁立雪,等.支气管败血波氏杆菌PCR检测方法的建立和应用[J].山东农业大学学报(自然科学版),2009,40(01):70-74.
[11]邸海波,楚常欢,宋苍浩,等.支气管败血波氏杆菌、多杀性巴氏杆菌和副猪嗜血杆菌同时检测的PCR方法的建立[J].畜禽业,2010,(05):57-59.
[12]熊富强,王芳,恽时锋,等.兔支气管败血波氏杆菌双夹心ELISA检测方法的建立[J].江苏农业学报, 2010, 26(01):120-125.
[13]徐婷婷.荧光定量PCR技术应用综述[J].畜禽业,2010,(10):48-50.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |