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Method for detecting and identifying plasmodium sperozoites in mosquito medium

A Plasmodium and identification method technology, applied in biochemical equipment and methods, resistance to vector-borne diseases, measurement/inspection of microorganisms, etc., can solve the problem of high cost of Taqman probes, low cost of Plasmodium sporozoites, inability to carry out parasite detection Specific identification and other issues, to achieve the effect of improving sensitivity and specificity, and low cost

Inactive Publication Date: 2011-08-17
JIANGSU INST OF PARASITIC DISEASES
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AI Technical Summary

Problems solved by technology

Existing abroad adopts the report of fluorescent quantitative PCR technique to detect Plasmodium falciparum infection in the mosquito body at present, and its shortcoming is that can't carry out insect species identification (Bell, A.S.L.C.Ranford-Cartwright.A real-time PCR assay forquantifying Plasmodium falciparum infections in the mosquito vector[J ]. IntJ Parasitol, 2004, 34 (7): 795-802); Bass et al. (Bass, C., D.Nikou, A.M.Blagborough, et al. high-throughput assay with existing methods [J]. Malar J, 2008, 7: 177) established Taqman probe fluorescent quantitative PCR technology for detection of malaria parasites in mosquitoes and identification of species, but due to the high cost of Taqman probes , not suitable for large sample detection and field application
At present, there is no report on the detection and identification of Plasmodium sporozoites in four types of mosquitoes using the low-cost and easy-to-operate fluorescent quantitative PCR technology based on SYBR Green I dye method

Method used

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  • Method for detecting and identifying plasmodium sperozoites in mosquito medium
  • Method for detecting and identifying plasmodium sperozoites in mosquito medium
  • Method for detecting and identifying plasmodium sperozoites in mosquito medium

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Embodiment 1

[0040] 1. Design primers:

[0041] According to the sequence characteristics of the 18S small subunit ribosomal rDNA of four human malaria parasites, a pair of primers were designed on the genus-specific regions on both sides of the species-specific region. For sequence comparison, see figure 1 . The primer sequences are as follows:

[0042] 18S-qF: 5'ATAACGAACGAGATCTTAACCT-3'

[0043] 18S-qR: 5'-ATCGTTCCTCTAAAGAAGCTTTA-3'.

[0044] 2. Extraction of Anopheles Plasmodium genomic DNA:

[0045] a) Anopheles cephalothorax is placed in a 1.5ml centrifuge tube;

[0046] b) Add 50 μl 1×PBS to the centrifuge tube described in step a), grind with a glass grinding rod for 5-10 minutes, then add 100 μl Buffer ATL;

[0047] c) Add 20 μl proteinase K to the centrifuge tube described in step b), shake at 56° C. for 3 hours;

[0048] d) Add 200 μl Buffer Al to the centrifuge tube described in step c), vibrate for 15 seconds, and place at 70°C for 10 minutes;

[0049] e) Add 200 μl of...

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Abstract

The invention discloses a method for detecting and identifying plasmodium sperozoites in a mosquito medium. The method comprises the following steps of: 1) designing a pair of primers; 2) extracting a plasmodium sperozoite genome deoxyribonucleic acid (DNA) in anopheles serving as a medium; 3) performing amplification on the plasmodium sperozoite genome DNA in the mosquito medium by a fluorescent quantitative polymerase chain reaction (PCR); and 4) melting an amplification product to obtain a melting curve, and comparing with positive control and negative control. The method has the advantages that: through the method for detecting and identifying plasmodium sperozoites in the mosquito medium and application thereof, plasmodia in the mosquito medium can be quantitatively detected, and can be qualitatively identified according to plasmid DNA standard substances of four constructed human plasmodia (which are shown as the figure 2), the sensibility and specificity of the detection and identification technology are improved, and compared with the prior art, the method is simple, convenient and quick and low in cost, and is suitable for evaluating the malaria control effect and monitoring malaria.

Description

technical field [0001] The present invention relates to a method for detecting and identifying Plasmodium sporozoites in mosquito media. Background technique [0002] Malaria is a vector-borne infectious disease that seriously endangers human health. Every year, 300-500 million people get sick and at least 1 million people die from malaria. The World Health Organization (WHO) has listed malaria, AIDS, and tuberculosis as three major global public health problems. There are four species of Plasmodium that cause malaria in humans: Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale. [0003] Anopheles mosquito is the only vector of malaria, and vector control measures are of great significance to the realization of global control and elimination of malaria goals (Greenwood, B.M. Control to elimination: implications for malaria research [J]. Trends Parasitol, 2008, 24 (10): 449-454; WHO Study Group. Malaria vector control and personal protection ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06G01N21/64
CPCY02A50/30
Inventor 高琪刘耀宝曹俊周华云
Owner JIANGSU INST OF PARASITIC DISEASES
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