Rui-Qing Zhang1, Zheng Li2, Gui-Xia Li3, Yan-Qing Tie4, Xin-Na Li5, Yuan Gao6, Qing-Xia Duan7, Le Wang8, Li Zhao9, Guo-Hao Fan10, Xue-Ding Bai11, Rui-Huan Wang12, Zi-Wei Chen13, Jin-Rong Wang14, Yong Wu15, Meng-Chuan Zhao16, Zhi-Shan Feng17, Ji Wang18, Xue-Jun Ma19. 1. Hebei Medical University, Shijiazhuang, 050031, Hebei, China; NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China; Hebei General Hospital, Shijiazhuang, 050051, Hebei, China. Electronic address: hebmuzhangruiqing@163.com. 2. Hebei General Hospital, Shijiazhuang, 050051, Hebei, China. Electronic address: 1226079314@qq.com. 3. Children's Hospital of Hebei Province, Shijiazhuang, 050031, Hebei, China. Electronic address: 13832179762@139.com. 4. Hebei General Hospital, Shijiazhuang, 050051, Hebei, China. Electronic address: 330614484@qq.com. 5. NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: zhaoyanghefeng@126.com. 6. Hebei Medical University, Shijiazhuang, 050031, Hebei, China; NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: 1842374876@qq.com. 7. Hebei Medical University, Shijiazhuang, 050031, Hebei, China; NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: 15227821073@163.com. 8. Children's Hospital of Hebei Province, Shijiazhuang, 050031, Hebei, China. Electronic address: luka_wl@163.com. 9. Children's Hospital of Hebei Province, Shijiazhuang, 050031, Hebei, China. Electronic address: zhaoli0226@163.com. 10. NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: 1059987716@qq.com. 11. Tangshan Gongren Hospital, Tangshan, 063000, China. Electronic address: 1120008531@qq.com. 12. Hunan Provincial Center for Disease Control and Prevention, Hunan, 410005, China. Electronic address: 15286855786@163.com. 13. The Third Xiangya Hospital of Central South University, Hunan, 410013, China. Electronic address: 852697879@qq.com. 14. Hebei Medical University, Shijiazhuang, 050031, Hebei, China; NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: 15232728122@163.com. 15. Health Gene Technologies, Ningbo, 315040, China. Electronic address: 277607480@qq.com. 16. Children's Hospital of Hebei Province, Shijiazhuang, 050031, Hebei, China. Electronic address: zhaomengchuan1989@163.com. 17. Hebei Medical University, Shijiazhuang, 050031, Hebei, China; Hebei General Hospital, Shijiazhuang, 050051, Hebei, China. Electronic address: 15131129999@139.com. 18. NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: wangji19871229@sina.com. 19. NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address: maxj@ivdc.chinacdc.cn.
Abstract
OBJECTIVES: Bordetella pertussis is a highly contagious respiratory agent and is the causative pathogen of pertussis, which primarily affects children. Current diagnostic techniques for this pathogen have a variety of limitations including a long culture time, low bacterial load, and lack of specificity. METHODS: This article reports the development of a one-tube nested quantitative real-time PCR assay using the locked nucleic acid (LNA) technique (LNA-OTN-q-PCR), targeting the BP485 gene and using a simple inexpensive extraction method. A total of 130 clinical samples from patients with clinically suspected pertussis, collected from the Children's Hospital of Hebei, China, were tested by LNA-OTN-q-PCR assay. RT-PCR and two-step semi-nested PCR assays were performed in parallel for comparison. RESULTS: Only strains of B. pertussis were identified as positive, whereas all of the remaining strains were appropriately identified as negative by the LNA-OTN-q-PCR assay. A single copy per reaction can be detected by the LNA-OTN-q-PCR assay. Additionally, the sensitivity of this method was 100 times that of the RT-PCR assay (100 copies per reaction). Sixty-three of the 130 clinical samples were detected positive by LNA-OTN-q-PCR assay; in contrast, RT-PCR was able to detect only 41 positive samples. Following this, all 63 samples were positively identified by two-step semi-nested PCR. Compared with the two-step semi-nested PCR assay, both the specificity and sensitivity of the LNA-OTN-q-PCR assay using purified DNA and crude extract were 100%. CONCLUSIONS: This assay was able to detect B. pertussis infection with high sensitivity and specificity. This test shows great potential as a promising technique to detect B. pertussis in both clinical laboratories and public health settings.
OBJECTIVES: Bordetella pertussis is a highly contagious respiratory agent and is the causative pathogen of pertussis, which primarily affects children. Current diagnostic techniques for this pathogen have a variety of limitations including a long culture time, low bacterial load, and lack of specificity. METHODS: This article reports the development of a one-tube nested quantitative real-time PCR assay using the locked nucleic acid (LNA) technique (LNA-OTN-q-PCR), targeting the BP485 gene and using a simple inexpensive extraction method. A total of 130 clinical samples from patients with clinically suspected pertussis, collected from the Children's Hospital of Hebei, China, were tested by LNA-OTN-q-PCR assay. RT-PCR and two-step semi-nested PCR assays were performed in parallel for comparison. RESULTS: Only strains of B. pertussis were identified as positive, whereas all of the remaining strains were appropriately identified as negative by the LNA-OTN-q-PCR assay. A single copy per reaction can be detected by the LNA-OTN-q-PCR assay. Additionally, the sensitivity of this method was 100 times that of the RT-PCR assay (100 copies per reaction). Sixty-three of the 130 clinical samples were detected positive by LNA-OTN-q-PCR assay; in contrast, RT-PCR was able to detect only 41 positive samples. Following this, all 63 samples were positively identified by two-step semi-nested PCR. Compared with the two-step semi-nested PCR assay, both the specificity and sensitivity of the LNA-OTN-q-PCR assay using purified DNA and crude extract were 100%. CONCLUSIONS: This assay was able to detect B. pertussis infection with high sensitivity and specificity. This test shows great potential as a promising technique to detect B. pertussis in both clinical laboratories and public health settings.