疟疾蚊媒监测多重PCR方法的建立

doi:10.12140/j.issn.1000-7423.2022.02.005

摘要/Abstract

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、190~320 bp）,吸食人血后的中华按蚊样品为3个条带（519、432、190~320 bp）,未吸血或吸食畜血的中华按蚊样品为2个条带（432、190~320 bp）,吸人血的非中华按蚊样品为2个条带（519、190~320 bp）,未吸血或吸食畜血的非中华按蚊样品为1个条带（190~320 bp）。特异性检测结果显示,11种其他寄生虫DNA样品结果均为阴性;对畜圈周围采集的野生中华按蚊样品和室内吸食人血的蚊虫样品检测结果显示,人血引物的特异性良好。结论本研究建立的疟疾蚊媒多重PCR检测方法,1次检测可同时获取蚊种鉴定、人血指数和疟原虫感染等多方面信息,敏感性高、特异性好。

关键词: 疟原虫, 蚊媒监测, 多重PCR

Abstract:

Objective 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 4 primer 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 25 times. 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, respectively. After optimization of multiplex PCR, the results showed that the assay has the highest sensitivity for P. vivax, with the detection limit of 10.7 parasite/μl blood. The assay had the lowest sensitivity for P. malariae with 133.3 parasite/μl blood detection limit. The detection limit for P. ovale and P. falciparum were 15.0 and 34.0 parasite/μl blood, respectively. The average detection limit was 48.25 parasite/μl blood. For An. sinensis mosquitos, simulated risk infection samples shows four bands (662-717, 519, 432, 190-320 bp), three bands (519, 432, 190-320 bp) in mosquitos which had human blood-meal, and two bands (432, 190-320 bp) in mosquitos which did not have blood-meal. Two bands (519, 190-320 bp) were shown in non-An. sinesis mosquito samples which had human blood-meal, only one band (190-320 bp) was shown from mosquitos which did not have blood-meal. For specificity, results from 11 other parasites samples are negative. Wild mosquito samples from An. sinensis around the corral and indoor human blood-fed samples showed good primer specificity for human blood. Conclusion The multiplex PCR method established in this study for malaria vector mosquito could provide multiple information on mosquito species identification, human blood index and malaria parasite infection simutanously in one assay, showing high sensitivity and specificity.

Key words: Malaria parasites, Mosquito vector surveillance, Multiplex PCR

中图分类号:

R382.31

江莉, 张耀光, 刘红霞, 王真瑜, 朱民, 吴寰宇. 疟疾蚊媒监测多重PCR方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(2): 159-167.

JIANG Li, ZHANG Yao-guang, LIU Hong-xia, WANG Zhen-yu, ZHU Min, WU Huan-yu. Establishment of multiplex PCR for malaria-transmitting vector surveillance[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(2): 159-167.

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基因来源 Source of genes	特异性引物序列（5′→3′） Specific primer sequences (5′→3′)	产物长度/bp Product length/bp	参考序列 Reference sequence
疟原虫属 Plasmodium	F: GCGGTAATTCCAGCTCCAATAG R: TCTTTTAACTTTCTCGCTTGCGC	662~717	M19173
人类 Homo sapiens	F: CGAGAGTTCTCTGGAAGAATTGA R: TGATAGCCTGGAAGTGACAAAAT	519	X68195
中华按蚊 Anopheles sinensis	F: GAACCTACCATGACGTACACATAC R: GTTGTCCAGCCCGCTAACAT	432	KU497636
蚊通用 Universal primer for mosquitoes	F: CCAAGAAGTCTATCTTGCGCG R: TGCACGTCAGAATTGCTTCG	190~320	JN975422

疟原虫种类 Parasite species	原虫密度/个虫·（μl血）^-1 Parasite count/No.·(μl blood)^-1	最高稀释度 Highest dilution	最低原虫密度/个虫·（μl血）^-1 Lowest parasite density/No.·(μl blood)^-1	平均检出限/个虫·（μl 血）^-1 Average detection limit/No.·(μl blood)^-1
间日疟原虫 Plasmodium vivax	6 857	1 ∶ 640	10.7	48.25
卵形疟原虫 P. ovale	9 627	1 ∶ 640	15.0
恶性疟原虫 P. falciparum	27 200	1 ∶ 800	34.0
三日疟原虫 P. malariae	10 666	1 ∶ 80	133.3