实时荧光定量PCR检测鸭疫里默氏杆菌方法的建立和应用

Development and application of a fluorogenic quantitative PCR assay for detecting Riemerella anatipestifer DNA

根据我们实验室发现的鸭疫里默氏杆菌(RA)荚膜多糖蛋白基因序列(DQ151838),设计和合成荧光定量PCR引物和探针,建立了检测RA的实时荧光定量PCR方法。该法的表达式Y=-3.416X+39.492,相关系数1.000,PCR循环效率96.2%,DNA拷贝数在100～108范围内检测曲线有良好的线性关系。该法的最适Mg2+、引物和探针浓度分别为4～5mmol/L、0.16～0.2μmol/L和0.16μmol/L,最低能够检测到RA的DNA模板为2.0copies/μL。1/10半数致死量的RA人工皮下注射感染7日龄樱桃谷鸭后2h即可在心、肝、肺、胸腺、食管中检测到RA核酸;感染后8h即可在心、肝、脾、肺、肾、脑、胰、食管、气管、胸腺、法氏囊、十二指肠、直肠、血液和咽喉拭子检测到RA核酸,36～120h是RA在感染雏鸭除了咽喉、食管和气管各个组织器官繁殖数量最高峰期,其后逐渐下降。RA人工感染致死雏鸭的心、肝、脑RA分离和FQ-PCR检测符合率为100%。对临床送检具有典型鸭疫里默氏杆菌病临床症状和病理变化的死亡雏鸭的肝脏、心血和脑组织的FQ-PCR检测的阳性率均为100%,而RA分离的阳性率分别只有74.3%(26/35)、82.9%(29/35)和91.4%(32/35)。FQ-PCR可用于鸭疫里默氏杆菌感染的快速诊断、流行病学调查和鸭产品的检疫等。

A rapid Fluorogenic quantitative PCR（FQ-PCR） assay was established using the capsular polysaccharide protein gene of Riemerella anatipestifer（DQ151838）. The assay contains a pare of primers and an internal dual-labeled fluorogenic probe. The equation of the assay was Y=-3. 416X＋39. 492, and the correlation coefficient was 1. 000 and PCR efficiency of crying reached up to 96.2 %. There was an excellent linear relation during the DNA concentration from 2.0 to 2.0× 10^8 copies. The best reaction condition was 4.0 to 5.0mmol/L for Mg^2＋ , 0.16 to 0.2 μmol/L for primers and 0.16 μmol/L for probe. The sensitivity of the assay was 2.0 copies/μL of Riemerella anatipestifer （RA） DNA. The negative results were obtained for detecting Escherichia Genus, Salmonella Genus and Staphylococcus Genus. Seven-day-old duckling infected with 1/10 LD50 RA, and the results indicated that RA-DNA could be detected in heart, liver, lung, thymus, esophagus 2 hours after inoculation, and could be detected in heart, liver, lung, thymus, esophagus,kidney, brain, pancreas, esophagus, trachea, bursal, duodenum, rectum, blood and pharyngeal swab 8 hours post infection （PI）. The amounts of RA-DNA reached to peak 36-120 hours PI in all detected organs except esophagus, trachea and pharyngeal, and then went down. The positive coincidence rate when comparing RA isolation with FQ-PCR to detect the samples of heart, liver and brain from died duckl-ing experimental infected with RA was 100 percent. When detecting the samples of heart, liver and brain from dead duckling with RA isolation and FQ-PCR, the positive rate by isolation are 74.3%（26/ 35） .82.9%（29/35） and 91.4%（32/35） respectively, but 100% positive for all samples by FQ-PCR. FQ- PCR can be used for the epidemiological investigation, rapid detection and duck product quarantine for RA infection.