Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCR
Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCR
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DOI：
10.1051/vetres:2005035
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发表时间：
2005-09-01
影响因子：
4.4
通讯作者：
Vanrompay, D
中科院分区：
文献类型：
--
作者：
Geens, T;Dewitte, A;Vanrompay, D
建立了鹦鹉热嗜衣原体rDNA核糖体间隔区和Cp基因型特异性实时荧光PCR方法。鹦鹉热外膜蛋白A（ompA）基因。以SYBR Green为基础的种特异性实时荧光PCR检测Cp。鹦鹉热基因型A至F，以及最近发现的E/B基因型。基因型特异性实时荧光定量PCR技术可通过TaqMan探针区分C、D、F基因型。基因型A、B和E不能通过简单的TaqMan探针相互区分。为此，必须在TaqMan探针旁边使用非荧光竞争寡核苷酸。基因型E/B只能通过使用小沟结合物（MGB）探针来检测。这两种实时PCR检测方法均可重复、灵敏（10 rDNA或ompA拷贝/μ L DNA提取物）和特异性检测Cp。鹦鹉热DNA将基因型特异性实时PCR与使用5个Cp的ompA测序和ompA限制性片段长度多态性（RFLP）分析进行比较。鹦鹉热野外分离株（99、61/8、7344/2、8615/1和7778 B15）均由两种不同的基因型组成。目前开发的实时PCR检测用于兽医学校和火鸡农场的案例研究。在兽医学院，CP。在所有五组火鸡中检测到鹦鹉热基因型D、E/B和F感染，以及一名照顾所有这些火鸡的兽医。在火鸡农场，两个CP的存在。在一只随机选择的火鸡中证实了鹦鹉热基因型B感染波，第一波在6周龄，第二波在12周龄。
A Chlamydophila psittaci species-specific real-time PCR targeting the rDNA ribosomal spacer was developed as well as a genotype-specific real-time PCR targeting the Cp. psittaci outer membrane protein A ( ompA) gene. The SYBR Green-based species-specific real-time PCR detected Cp. psittaci genotypes A to F, and the recently discovered E/B genotype. The genotype-specific real-time PCR could easily distinguish genotypes C, D, F by use of TaqMan probes. Genotypes A, B and E could not be distinguished from each other by simply using TaqMan probes. For this purpose, non-fluorescent competitor oligonucleotides, had to be used next to the TaqMan probes. Genotype E/B could only be detected by use of a minor groove binder (MGB) probe. Both real-time PCR assays allowed reproducible, sensitive ( 10 rDNA or ompA copies/mu L DNA extract) and specific detection of Cp. psittaci DNA. The genotype-specific real-time PCR was compared to ompA sequencing and ompA restriction fragment length polymorphism ( RFLP) analysis using five Cp. psittaci field isolates ( 99, 61/8, 7344/2, 8615/1 and 7778B15) each consisting of two different genotypes. The currently developed real-time PCR assays were used in a case study on a veterinary school and a turkey farm. In the veterinary school, Cp. psittaci genotypes D, E/B and F infection were detected in all five groups of turkeys, and one veterinarian who was taking care of all these turkeys. On the turkey farm, the presence of two Cp. psittaci genotype B infection waves was demonstrated in one randomly selected turkey, the first wave at the age of 6 weeks, and the second at the age of 12 weeks.