CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES ›› 2022, Vol. 40 ›› Issue (2): 159-167.doi: 10.12140/j.issn.1000-7423.2022.02.005
• ORIGINAL ARTICLES • Previous Articles Next Articles
JIANG Li*(), ZHANG Yao-guang, LIU Hong-xia, WANG Zhen-yu, ZHU Min, WU Huan-yu
Received:
2021-07-20
Revised:
2021-10-11
Online:
2022-04-15
Published:
2022-04-15
Contact:
JIANG Li
E-mail:jiangli@scdc.sh.cn
Supported by:
CLC Number:
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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URL: https://www.jsczz.cn/EN/10.12140/j.issn.1000-7423.2022.02.005
Table 1
Fragments amplified by specific primer sequences
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 |
Fig. 1
The result of multiple PCR detecting different mosquito species and simulated positive samples with Plasmodium falciparum infection M: DNA marker; 1: Aedes albopictus; 2: Culex pipiens pallens; 3: Anopheles sinensis; 4: Anopheles sinensis simulated positive sample A2.5(containing parasite density of 2 662.5 per microliter of P. falciparum); 5: Culex pipiens pallens simulated positive sample C2.5(containing parasite density of 2 662.5 per microliter of P. falciparum).
Fig. 2
Effects of primer concentration on different gene amplification efficiencies M: DNA marker; A: Each primer equally mixed in the primer Mix; B, C: Mosquito genes primer diluted 10 times and 20 times respectively in the primer Mix; D: Anopheles sinensis simulated positive sample A2.5 (containing parasite density of 2 662.5 per microliter of P. falciparum); E: Culex pipiens pallens simulated positive sample C2.5 (containing parasite density of 2 662.5 per microliter of P. falciparum); 1, 2: Culex pipiens pallens simulated positive sample C5 and C10 (containing parasite density of 5 325 and 10 650 respectively per microliter of P. falciparum); 3-5: Dosage of Plasmodium primers with 1, 1.5 and 2 μl, respectively.
Fig. 3
Sensitivity analysis of mosquito vector multiplex PCR reaction system M: DNA marker; 1-5: P. vivax patient blood sample diluted at 1 ∶ 40-1 ∶ 640; 6-10: P. ovale patient blood sample diluted at 1 ∶ 160-1 ∶ 2 560; 11-15: P. falciparum patient blood sample diluted at 1 ∶ 100-1 ∶ 1 600; 16-20: P. malariae patient blood sample diluted at 1 ∶ 20-1 ∶ 320.
Table 2
Sensitivity of parasite density tested by mosquito vector multiplex PCR
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 |
Fig. 4
The specificity of mosquito vector multiplex PCR detecting other parasite DNA M: DNA marker; P: Anopheles sinensis simulated positive control(containing DNA of Anopheles sinensis and P. falciparum with parasite density at 2 662.5/μl); N: Negative control of Anopheles sinensis (DNA of Anopheles sinensis); B: Blank control; 1-12: DNA from Entamoeba histolytica, Giardia lamblia, Cryptosporidium, Babesia microti, Leishmania donovani, Plasmodium falciparum, Toxoplasma gondii, Schistosoma japonicum, Paragonimus westermani, Ascaris lumbricoides, Taenia saginata, T. solium.
Fig. 5
Wild mosquito samples tested by mosquito vector multiplex PCR M: DNA marker; 1: Culex pipiens pallens simulated positive sample C2.5 (containing parasite density at 2 662.5/μl of P. falciparum); 2: Anopheles sinensis simulated positive sample A2.5 (containing parasite density at 2 662.5/μl of P. falciparum); 3: Anopheles sinensis simulated positive sample A2.5 Pv(containing parasite density at 250/μl of P. vivax); 4: Mosquito sample 1 collected indoors; 5: Mosquito sample 2 collected indoors; 6: P. vivax blood sample dilution 10-4; 7-22: Wild Anopheles sinensis mosquito samples; N: Negative control(DNA of Schistosoma japonicum); B: Blank control; P: Anopheles sinensis simulated positive control(containing DNA of P. falciparum).
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