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Multiple polymerase chain reaction (PCR) kit and method for detecting mosquito-borne pathogens

A technology of pathogens and kits, which is applied in the genetic detection of mosquito-borne pathogens and the field of multiple PCR kits for detection of mosquito-borne pathogens, which can solve the problems of low sensitivity, inability to detect multiple mosquito pathogens at the same time, and high cost

Inactive Publication Date: 2011-02-23
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] The object of the present invention is to provide a kind of multiplex PCR test kit and detection method that detects mosquito-borne pathogen, described this multiplex PCR kit that detects mosquito-borne pathogen and detection method will solve the problem of detecting mosquito-borne pathogen in the prior art The method has low sensitivity, high cost, and the technical problems of being unable to detect multiple pathogens carried by mosquitoes at the same time

Method used

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  • Multiple polymerase chain reaction (PCR) kit and method for detecting mosquito-borne pathogens
  • Multiple polymerase chain reaction (PCR) kit and method for detecting mosquito-borne pathogens
  • Multiple polymerase chain reaction (PCR) kit and method for detecting mosquito-borne pathogens

Examples

Experimental program
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Effect test

Embodiment 1

[0089] A kind of multiplex PCR kit for detecting mosquito-borne pathogens of the present invention contains following primers:

[0090] 1) Detection of primers for the first round of PCR of flaviviruses

[0091] The sequence of the upstream primer is shown in SEQ ID NO: 1,

[0092] The sequence of the downstream primer is shown in SEQ ID NO: 2;

[0093] 2) Detect the primers of the second round PCR of flavivirus

[0094] The sequence of the upstream primer is shown in SEQ ID NO: 3,

[0095] The sequence of the downstream primer is shown in SEQ ID NO: 4;

[0096] 3) Primers for the first round of PCR to detect Plasmodium

[0097] The sequence of the upstream primer is shown in SEQ ID NO: 5,

[0098] The sequence of the downstream primer is shown in SEQ ID NO: 6;

[0099] 4) Primers for the second round of PCR detection of Plasmodium

[0100] The sequence of the upstream primer is shown in SEQ ID NO: 7 or shown in SEQ ID NO: 8,

[0101] The sequence of the downstream pri...

Embodiment 2

[0127] The present invention also provides a method for detecting mosquito-borne pathogens, including a step of extracting mosquito nucleic acid, a step of amplifying the extracted nucleic acid on a PCR amplification instrument, and a step of amplifying the amplified product The step of electrophoresis detection also includes the step of sequencing to determine the specific species of mosquito-borne pathogens.

[0128] Further, in a step of amplifying the extracted nucleic acid on a PCR amplification instrument,

[0129] 1) Detection of primers for the first round of PCR of flaviviruses

[0130] The sequence of the upstream primer is shown in SEQ ID NO: 1,

[0131] The sequence of the downstream primer is shown in SEQ ID NO: 2;

[0132] 2) Detect the primers of the second round PCR of flavivirus

[0133] The sequence of the upstream primer is shown in SEQ ID NO: 3,

[0134] The sequence of the downstream primer is shown in SEQ ID NO: 4;

[0135] 3) Primers for the first r...

Embodiment 3

[0147] Example 3 The steps for detecting mosquitoes carrying Plasmodium (Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale, Plasmodium malariae and Filaria malayi) are described as follows:

[0148] a. Extraction of nucleic acid: Take a single mosquito (Anopheles stepheni) and grind it into a 1.5ml centrifuge tube until it is completely ground into powder. Use Tiangen DNA Extraction Kit DP304 (Beijing Tiangen) to extract DNA according to the instructions.

[0149] b.PCR amplification:

[0150] The first round of PCR conditions

[0151] 10×Buffer 5μL

[0152] dNTP (2.5mM) 4μL

[0153] BSA (5mg / ml) 1μL

[0154] FP (10μM) 1μL each (filariae Plasmodium first-round PCR primers)

[0155] 1 μL each of RP (10 μM) (the first round of PCR primers for filarial plasmodium)

[0156] DNA template 1.5 μL

[0157] EXTaq 0.4μL (2U)

[0158]wxya 2 O 34.1 μL

[0159] PCR cycling parameters

[0160] 1. 94℃ 4Min

[0161] 2. 94℃ 25Sec

[0162] 3. 56℃ 20Sec ...

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Abstract

The invention provides a multiple polymerase chain reaction (PCR) kit for detecting mosquito-borne pathogens. The kit comprises six pairs of specific primers. The invention also provides a method for detecting the mosquito-borne pathogens. Electrophoresis is performed on a PCR-amplified product. Whether pathogens, such as encephalitis B virus, dengue fever virus, yellow fever virus, plasmodium falciparum, plasmodium vivax, plasmodium knowlesi, plasmodium ovale, plasmodium malariae, wuchereria malayi, wuchereria bancrofti and the like, exists or not is detected and identified according to the length of a PCR-amplified fragment. By the method, various reported mosquito-borne pathogens, and the yellow fever virus and west nile virus which come from other countries can be detected quickly, accurately and sensitively at the same time, and can be applied to the detection of various samples, such as mosquitoes, blood of patients, tissue fluid and the like. The invention provides a low-cost and high-efficient method for early monitoring and finding mosquito-borne disease prevalence for prevention and control work of mosquito-borne diseases in China.

Description

Technical field: [0001] The invention relates to the field of biological detection, in particular to genetic detection of mosquito-borne pathogens (Japanese encephalitis virus, dengue virus, malaria parasite, filarial), in particular to a multiplex PCR kit and detection method for detecting mosquito-borne pathogens. Background technique: [0002] The mosquito-borne diseases that are prevalent or have been prevalent in my country include malaria, filariasis, Japanese encephalitis, and dengue fever. Detection of mosquito-borne pathogens is one of the effective measures for early detection of epidemics and prevention of mosquito-borne diseases. [0003] Mosquitoes are the vectors of various infectious diseases such as malaria, filariasis, Japanese encephalitis, and dengue fever. For the monitoring of mosquito-borne diseases, most of the previous studies focused on the detection of a single pathogen, and the prevention and control of mosquito-borne diseases should be considered...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12Q1/68C12Q1/70
CPCY02A50/30
Inventor 周正斌朱淮民
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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