CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >
Establishment of multiplex PCR for malaria-transmitting vector surveillance
Received date: 2021-07-20
Revised date: 2021-10-11
Online published: 2022-04-15
Supported by
Project of Shanghai Scientific and Technological Innovation Action Plan(20DZ2200300);Fifth Round Three-year Action Plan on Key Subject of Shanghai Public Health System Construction(GWV-10.1-XK13)
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
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 . DOI: 10.12140/j.issn.1000-7423.2022.02.005
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