應用巢式聚合酶連鎖反應診斷豬隻鼻腔擦拭液之豬肺炎黴漿菌

Diagnosis of Mycoplasma Hyopneumoniae Infection from Swine Nasal Swabs via Nested Polymerase Chain Reaction

陳嘉蓉(Jia-Rong Chen) ； 邱明堂(Ming-Tang Chiou) ； 翁仲男(Chung-Nan Weng)

《臺灣獸醫學雜誌》 29卷1期 (2003/03) Pp. 59-65

https://doi.org/10.7009/TVJ.200303.0059

摘要

為早期診斷豬黴漿菌肺炎的引發病原—豬肺炎黴漿菌，本研究利用二組具物種專一性的引子行聚合酶連鎖反應來增幅16S rRNA基因片段。豬肺炎黴漿菌的DNA 在第一次及第二次的PCR增幅反應中各產生679bp及396bp的基因產物。分析此巢式PCR反應的敏感度顯示第一次PCR反應之偵測極限是1.2 x 10^4個豬肺炎黴漿菌，取第一次PCR反應的產物進行1000倍稀釋再行第二次PCR反應時敏感度增加1000倍，偵測極限是12個豬肺炎黴漿菌。另外，偵測豬肺炎黴漿菌DNA時若外加鼻腔擦拭液所純化的DNA並不會影響兩次PCR反應的敏感度。分析此巢式PCR反應的專一性顯示只有豬肺炎黴漿茵的DNA在兩次的增幅反應中各有不同片斷的基因產物合成，M. flocculare之DNA在第一次PCR反應中產生679 bp 的基因產物，第二次PCR反應則無基因產物合成。M. hyorhinis之DNA在兩次的PCR反應中均無基因產物合成。因此此二組引子可用於區分豬隻中常見的三種黴漿菌。應用巢式PCR反應檢測15個SPF豬鼻腔擦拭液的檢體，結果顯示第一次PCR反應無法偵測到黴漿菌，但是經第二次PCR反應後卻發現有7個檢體感染豬肺炎黴漿菌。應用於檢測豬肺臟檢體發現6個SPF豬肺臟檢體在第一次PCR反應可偵測至2個肺臟感染黴漿菌，若經第二次PCR反應則增加5個檢體感染豬肺炎黴漿菌。總之，經兩次增幅反應的巢氏PCR反應其靈敏度高於傳統的一次PCR增幅反應，因此可應用於檢測雜菌污染程度較高且黴漿菌存在數量較少之鼻腔擦拭液檢體。

關鍵字

豬肺炎黴漿菌 ； 鼻腔擦拭液 ； 巢氏聚合酶連鎖反應

並列摘要

For early diagnosis of Mycoplasma hyopneumoniae infection, the causative agent of mycoplasmal pneumonia in swine, a nested PCR was performed in this study using 2 species-specific sets of primers to amplify two gene segments of the 16S rRNA gene. Amplification of M. hyopneumoniae DNA in the first and second PCR stages produced 679 bp and 396 bp gene products, respectively. Sensitivity assay showed that the detection limit in the first PCR stage was 1.2 x 10^4 M. hyopneumoniae organisms. The PCR product from the first stage was then 1000-fold diluted and used as template in the second stage. The sensitivity of the second PCR stage was markedly increased for 1000-fold and the detection limit was 12 genome equivalents of M. hyopneumoniae. Moreover, the sensitivity of the nested PCR in amplifying M. hyopneumoniae DNA was not influenced by the DNA purified from nasal swabs of non-M. hyopneumoniae infected swine. The nested PCR performed in this study was found to be specific for diagnosis of M. hyopneumoniae. Since M. flocculare DNA was amplified only in the first PCR stage and M. hyopneumoniae DNA was not influenced via the two-stage PCR, the 2 sets of primers could then be used to differentiate 3 Mycoplasma species commonly found in pigs. Application of the nested PCR to detect 15 nasal swabs from SPF pigs, it showed that none of the nasal swabs displayed a positive result in the first PCR stage, compared to positive results for 7 of the 15 nasal swabs in the second stage. Lung tissues from 6 SPF pigs were also examined for M. hyopneumoniae infection via nested PCR, the results revealed that 2 samples displayed positive results in the first stage, while 5 out of 6 gave positive results in the second stage. Taken together, the sensitivity of nested PCR via two amplification stages was superior to one amplification stage conventionally used and could be applied in detecting low quantities of Mycoplasma in more contaminated nasal cavity.

並列關鍵字

Mycoplasma hyopneumoniae ； Nasal swabs ； Nested polymerase chain reaction