淋病奈瑟菌核酸检测试剂盒国家参考品的制备  

Preparation of national reference for detection kit of neisseria gonorrhoeae nucleic acid

作　　者：王春娥[1] 卢旭[1] 刘茹凤[1] 石继春[1] 李红[1] 陈琼[1] 唐静[1] 陈翠萍[1] 叶强[1] 

机构地区：[1]中国食品药品检定研究院卫生部生物技术产品检定方法及其标准化重点实验室,北京100050

出　　处：《微生物学免疫学进展》2016年第5期21-25,共5页Progress In Microbiology and Immunology

基　　金：国家科技基础条件平台国家微生物资源平台奖补经费资助项目(NIMR 2015-2)

摘　　要：目的：制备淋病奈瑟菌（ Neisseria gonorrhoeae,NG）核酸检测试剂盒国家参考品。方法分别将10株NG和10株非NG参考菌株在各自适宜的培养基和温度下培养,收获新鲜无污染培养物,用比浊法和显微计数法将不同的培养物制备成10份NG菌悬液阳性参考品、5份精密性参考品和10份非NG菌悬液阴性参考品以及最低检出限参考品,然后利用5种不同来源的NG核酸检测试剂盒验证各种参考品,并考察参考品在不同处理条件下的稳定性。结果每株菌在各自适宜的培养基和温度下培养后,均生长良好,无杂菌污染。经5种试剂盒验证检测,10份阳性参考品的检测结果均为阳性,10份阴性参考品的检测结果均为阴性,精密性参考品检测结果为Ct值的CV均小于5.0%；5种试剂盒的最低检出限均能达到1000/mL,其中试剂盒B和E的最低检出限达100/mL。参考品在2-8℃放置7 d、37℃放置3 d的热稳定性及反复冻融5次以内的稳定性良好。结论制备的NG核酸检测试剂盒国家参考品的准确性、特异性及精密性均符合要求,可用于NG核酸检测试剂盒的质量评价。Objective To prepare the national reference for the detection kit of neisseria gonorrhoeae nucleic acid. Metho-ds All of 10 strains of neisseria gonorrhoeae and 10 strains of non-neisseria gonorrhoeae were cultured in a suitable media and temperature, then fresh and uncontaminated cultures were collected. The preparation of references was carried out in bacterial susponsion of both 10 positive reference of neisseria gonorrhoeae and 10 negtive reference of non-neisseria gonor-rhoeae, bacterial susponsion of 5 precision reference, and a bacterial susponsion of minimal detection limit reference by tur-bidimetry and microscopic counting. The references were verified by five PCR detection kits for neisseria gonorrhoeae from different companies, and evaluated by stability test under various conditions. Results Each from all strains grew well in its own suitable media and temperature, and the cultures were not contaminated. The detections showed correct results for 10 positive and 10 negative references, respectively, and the CV were all less than 5. 0% for tested results of precision by verification of the five PCR detection kits. A bacterial concentration of 1 000 /mL was determined by the five kits, and a concentration of 100 /mL determined by the kit No. B and E. The stability tests showed good results on the references after stored at 2-8 ℃ for 7 days,or 37 ℃ for 3 days,or 5 cycles of freezing-thawing, respectively. Conclusion The accuracy, specificity, reproducibility can met the relevant requirements for the prepared national references, which could be used in evaluation of quality control of PCR detection kit for neisseria gonorrhoeae nucleic acid.

关 键 词：淋病奈瑟菌 核酸 试剂盒 参考品 

分 类 号：R392-33[医药卫生—免疫学]