Güldemir Dilek, Akbaş Efsun, Nar Ötgün Selin, Tekin Alicem, Esen Berrin
Refik Saydam National Public Health Agency, Department of Communicable Diseases Research, Ankara, Turkey.
Mikrobiyol Bul. 2011 Oct;45(4):632-45.
Pertussis (whooping cough), caused by Bordetella pertussis is a severe, acute contagious disease of the respiratory system and it affects mostly children and also susceptible individuals of all ages. Although the conventional culture method used for diagnosis is highly specific, it has a lower sensitivity. Therefore, there is a need for a sensitive, specific and rapid method for diagnosis of pertussis. Polymerase chain reaction (PCR), introduced recently as a new approach for diagnosis of pertussis, has been shown to be more sensitive than culture method. Pertussis toxin gene (ptxA-Pr), insertion sequence genes (IS481 and IS1001), adenylate cyclase genes and structural porin and flagellin genes were chosen as targets for PCR, in different studies. This study aimed to develop and optimize a diagnostic inhouse PCR method by using primers specific for ptxA-Pr and IS481 gene regions. An in-house PCR method was developed by using primer pairs of PTp1/PTp2 specific for ptxA-Pr gene and PIp1/PIp2 specific for IS481 gene and DNAs of various bacterial reference strains. Throat samples obtained from 45 healthy individuals and B.pertussis reference strain with decreasing concentrations were mixed to constitute a group of "representative clinical samples" and used to test and optimize sensitivity and specificity of the method. The in-house PCR with PTp1/PTp2 primers showed a very high specificity but a low sensitivity with a value of 34.4 cfu/Rm (colony forming unit/reaction mixture). Whereas, the inhouse PCR with PIp1/PIp2 primers exhibited a low specificity due to cross-reactivity with B. Pertussis and B.bronchiseptica but much higher sensitivity with a value of 1.12 cfu/Rm. The experiments performed with the representative clinical samples yielded similar results. Simultaneously applied cultivation studies indicated the detection limit of the PCR method as 2 x 103 cfu/ml. Based on our results, the PCR targeting IS481 gene had high sensitivity while the PCR targeting ptxA-Pr gene had high specificity. It was concluded that, PCR method targeting the IS481 gene might be used for pre-diagnosis and then PCR for ptxA-Pr gene might be applied for the confirmation of B.pertussis in the molecular diagnosis of pertussis.
百日咳由百日咳博德特氏菌引起，是一种严重的急性呼吸系统传染病，主要影响儿童以及所有年龄段的易感人群。尽管用于诊断的传统培养方法具有高度特异性，但灵敏度较低。因此，需要一种灵敏、特异且快速的百日咳诊断方法。聚合酶链反应（PCR）作为一种新的百日咳诊断方法最近被引入，已被证明比培养方法更灵敏。在不同研究中，百日咳毒素基因（ptxA-Pr）、插入序列基因（IS481和IS1001）、腺苷酸环化酶基因以及结构孔蛋白和鞭毛蛋白基因被选为PCR的靶标。本研究旨在通过使用针对ptxA-Pr和IS481基因区域的特异性引物来开发和优化一种内部诊断PCR方法。通过使用针对ptxA-Pr基因的PTp1/PTp2引物对和针对IS481基因的PIp1/PIp2引物对以及各种细菌参考菌株的DNA，开发了一种内部PCR方法。从45名健康个体和浓度逐渐降低的百日咳博德特氏菌参考菌株获得的咽喉样本混合，构成一组“代表性临床样本”，用于测试和优化该方法的灵敏度和特异性。使用PTp1/PTp2引物的内部PCR显示出非常高的特异性，但灵敏度较低，值为34.4 cfu/Rm（菌落形成单位/反应混合物）。而使用PIp1/PIp2引物的内部PCR由于与百日咳博德特氏菌和支气管败血博德特氏菌的交叉反应而表现出低特异性，但灵敏度高得多，值为1.12 cfu/Rm。用代表性临床样本进行的实验产生了类似的结果。同时进行的培养研究表明PCR方法的检测限为2×103 cfu/ml。基于我们的结果，靶向IS481基因的PCR具有高灵敏度，而靶向ptxA-Pr基因的PCR具有高特异性。得出的结论是，靶向IS481基因的PCR方法可用于百日咳分子诊断中的预诊断，然后应用靶向ptxA-Pr基因的PCR进行百日咳博德特氏菌的确认。
