疟疾蚊媒监测多重PCR方法的建立  

Establishment of multiplex PCR for malaria-transmitting vector surveillance

作　　者：江莉[1] 张耀光[1] 刘红霞 王真瑜[1] 朱民[1] 吴寰宇 JIANG Li;ZHANG Yao-guang;LIU Hong-xia;WANG Zhen-yu;ZHU Min;WU Huan-yu(Shanghai Municipal Center for Disease Control and Prevention/Shanghai Institutes of Preventive Medicine,Shanghai 200336,China)

机构地区：[1]上海市疾病预防控制中心,上海市预防医学研究院,上海200336

出　　处：《中国寄生虫学与寄生虫病杂志》2022年第2期159-167,共9页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：上海市“科技创新行动计划”项目(20DZ2200300);上海市公共卫生体系建设三年行动计划第五轮重点学科(GWV-10.1-XK13)。

摘　　要：目的建立疟疾消除后高效的蚊媒监测多重PCR方法。方法设计疟原虫属、人血、中华按蚊和蚊通用4对特异性引物,与通用引物(5′-CGAGTCCTGCGGTCTCAAATT-3′)连接后,形成4对嵌合特异性引物。常规PCR确定各引物对的最佳退火温度和引物工作浓度,多重PCR优化4对引物混合后的最佳反应条件,引入模拟风险感染阳性蚊虫样品,建立敏感的多重PCR反应体系。检测4种疟疾(间日疟、卵形疟、恶性疟、三日疟)患者全血样品的原虫密度梯度稀释样品的各基因扩增情况,评估检测灵敏度。用溶组织内阿米巴、蓝氏贾第鞭毛虫、隐孢子虫、田鼠巴贝虫、杜氏利什曼原虫、刚地弓形虫、日本血吸虫尾蚴、卫氏并殖吸虫、蛔虫、牛带绦虫、猪带绦虫等11种其他寄生虫虫体DNA或感染样品评价该方法的检测特异性。用畜圈周围采集的野生中华按蚊样品,评价所建PCR方法的应用效果。结果优化后的多重PCR反应体系各组分含量(体积比)为:DNA模板10%、引物Mix10%、三蒸水30%,Taq酶预混液50%。引物Mix中,蚊通用、按蚊、人血和疟原虫引物的用量配比为1∶1.75∶3∶5。反应体系的最佳循环条件为:95℃5 min;94℃15 s,60℃20 s,72℃20 s,循环4次;94℃15 s;64℃20 s,72℃20 s,循环9次;94℃15 s,68℃20 s,72℃24 s,循环25次;72℃3 min;10℃5 min。疟原虫属扩增产物的长度为662~717 bp,人血为519 bp,中华按蚊为432 bp,蚊通用为190~320 bp。4种疟疾患者血样经优化后的多重PCR检测结果显示,该法对间日疟患者血样的灵敏度最高,检出最低原虫密度为10.7个虫/μl血;对三日疟患者血样的灵敏度最低,检出最低原虫密度为133.3个虫/μl血;对卵形疟和恶性疟患者血样的检出最低原虫密度分别为15.0和34.0个虫/μl血;检测灵敏度平均值为48.25个虫/μl血。检测模拟风险感染阳性蚊虫样品结果显示,中华按蚊模拟阳性样品显示为4个条带(662~717、519、432、190~320Objective To establish an highly efficient multiplex PCR method for surveillance of mosquito vector in malaria post-elimination stage.Methods Four pairs of specific chimeric primers were designed for amplifying specific genes of Plasmodium,human blood,Anopheles sinensis and mosquitoes,which was connected with universal primer(5′-CGAGTCCTGCGGTCTCAAATT-3′)respectively.Routing PCR reaction was used to determine the best annealing temperature and concentration of primers,and the multiplex PCR condition was optimized for the mixed 4primer pairs.The innovative design of simulated positive mosquito sample was introduced for establishing a sensitive multiplex PCR reaction system.The sensitivity was evaluated by detecting the parasite density using serially diluted samples from 4 Plasmodium species(P.falciparum,P.ovale,P.vivax and P.malariae)infected patient samples to check gene amplification.The specificity was evaluated by using a variety of other parasites’DNA or samples infected with other parasites,including Entamoeba histolytica,Giardia lamblia,Cryptosporidium,Babesia microti,Leishmania donovani,Toxoplasma gondii,Schistosoma japonicum,Paragonimus westermani,Ascaris lumbricoides,Taenia saginata,T.solium.Field evaluation was performed using wild An.sinensis samples collected from animal farms.Results The optimized reaction system for each component content(volume ratio)was:10% of DNA template,10% of primer Mix,30% of distilled water and 50% of Taq polymerase pre-mixed solution.The ratio of each primer in the mixture was 1∶1.75∶3∶5 for mosquitoes,An.sinensis,human blood and Plasmodium,respectively.The best circulation conditions for the system was as follows:95℃ for 5 min,94℃ for 15 s,60℃ for 20 s,and 72℃ for 20 s,circulation 4 times;94℃ for 15 s;64℃ for 20 s,72℃ for 20 s,circulation 9 times;94℃ for 15 s,68℃ for 20 s,72℃ for 20 s,circulating 25times.The length of the amplified products was 662-717 bp for Plasmodium,519 bp for human blood,432 bp for An.sinensis,190-320 bp for mosquitoes,

关 键 词：疟原虫 蚊媒监测 多重PCR 

分 类 号：R382.31[医药卫生—医学寄生虫学]