16S rDNA 유전자를 이용한 Bordetella 근연종으로부터 Bordetella pertussis의 감별 PCR
Discriminative PCR of Bordetella pertussisfrom closely related Bordetella species using16S rDNA Gene
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목 적 : 백일해의 원인균인 B. pertussis는 성장속도가 늦기 때문에 PCR 검출방법은 백일해 진단의 초기단계에서 유용하다. 현재 BP primer가 PCR 검출에 광범위하게 사용되나 BP primer는 최근에 B. holmesii와의 교차반응성이 보고되었다. 따라서 PCR 단계에서 B. pertussis와 B. holmesii의 감별이 필요하다. 이러한 이유로 본 논문에서는 16S rDNA sequence를 기반으로 하는 진단용 primer 를 새로 고안하였고, 그 효용성을 측정하였다. 재료 및 방법 : 특이적 PCR primer들은 다양한 Bordetella 속의 16S rDNA 유전자들의 정렬된 서열 집합체로부터 고안되었다. 고안된 primer의 특이도는 임상적으로 중요한 4개 Bordetella 종 (B. pertussis, B. holmesii, B. parapertussis, B. bronchiseptica)을 사용하여 평가하였다. 민감도 또한 평가하였고 그 결과를 BP primer와 비교하였다. 결 과 : 결과적으로, 새롭게 개발된 primer는 B. holmesii를 포함하는 다른 Bordetella 종들로부터 B. pertussis를 성공적으로 감별하였다. 민감도분석에서는 B. pertussis에 대한 16S-F2/16S-R1 primer의 검출한계는 5 pg의 genomic DNA와 105 cells/mL의 세포부유액까지 였다. 또한 임상검체에 대해서 BP primer set와 동일한 결과가 확인되었다. 결 론 : 본 연구에서 오직 B. pertussis에 대해 특이적인 PCR primer를 16S rDNA를 기반으로 하여 개발하였고, 이는 B. holmesii와의 교차반응성을 나타내지 않았다. 또한 본 연구에서 새롭게 개발된 primer의 임상검체에 대한 적용성도 확인되었다.
Abstract
Background : Polymerase-chain reaction (PCR) detection is useful to diagnosis of pertussis at initial stage because the growth rate of Bordetella pertussis (B. pertussis) is relatively slow. Currently, the primer set for the insertion sequence IS481 (BP primer) is used widely for PCR detection of B. pertussis. However, the cross-reactivity of BP primer set with Bordetella holmesii (B. holmesii) was reported recently. Therefore, discrimination of B. pertussis and B. holmesii is needed in PCR step. For this reason, we developed new primer sets based on 16S rDNA sequence for diagnostic use and estimated the efficiency of these new primer sets. Materials and Methods : The specific PCR primers were designed from the aligned sequence matrix of 16S rDNA genes of various Bordetella species. The specificity of designed primers were estimated using clinically important 4 Bordetella species, B. pertussis, B. holmesii, Bordetella parapertussis (B. parapertussis) and Bordetella bronchiseptica (B. bronchiseptica). The sensitivity to B. pertussis of designed primers was also estimated and compared with BP primer set. Results : As the results, the developed new primer set successfully distinguished B. pertussis and other Bordetella species containing B. holmesii. In the sensitivity assay, the detectable limits of 16S-F2/16S-R1 primer set for B. pertussis were revealed as 5 pg of genomic DNA and 105 cells/mL of cell suspension. In addition to these, identical results between BP with primer and new primer were obtained in clinical samples. Conclusion : In this study, the specific primer set for B. pertussis was developed based on 16S rDNA sequence and this primer set did not show cross-reactivity to B. holmesii. In addition to these, the applicability of this primer set to the clinical specimens was also confirmed.
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