Primer, method and kit for carrying out multiple tandem PCR gene disc detection on leishmania spp

A technology of Leishmania and Leishmania donovani, which is applied in the field of genetic engineering, can solve the problems of late diagnosis and treatment of cases, the lack of Leishmania detection reagents, etc., and achieve timely treatment of cases, rapid control of the epidemic, and detection results Clear and accurate results with high specificity and sensitivity

Pending Publication Date: 2022-01-07
四川国际旅行卫生保健中心(成都海关口岸门诊部)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difference in the epidemic situation at home and abroad, overseas laborers who are infected with Leishmaniasis face the situation that there are no Leishmania detection reagents when they enter the country, which has caused great difficulties in the later diagnosis and treatment of cases.

Method used

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  • Primer, method and kit for carrying out multiple tandem PCR gene disc detection on leishmania spp
  • Primer, method and kit for carrying out multiple tandem PCR gene disc detection on leishmania spp
  • Primer, method and kit for carrying out multiple tandem PCR gene disc detection on leishmania spp

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Primer Design

[0031] For four different types of Shmania, four positive recombinant plasmid templates containing ITS-1, ITS-2, P0, and RACK target gene fragments were constructed, and the plasmid DNA was extracted and stored in a -80°C ultra-low temperature refrigerator. Retrieve gene sequences (GenBankaccession no: AF306777, JN673394, JN673389, JN673385, AB175625, AY297458, AF499109 and M12691), use Primer Premier V5.0 and Oligo V6.22 for primer design and evaluation, and build nested primer pairs. For primer information, see Table 1.

[0032] Table 1. Primer information

[0033]

[0034]

Embodiment 2

[0035] Embodiment 2 multiplex tandem PCR

[0036] 1. Making gene discs

[0037] Take 11 μL of internal primers, add them into the amplification reaction plate, and adjust the concentration so that the final concentration reaches 0.4 μmol / L. Then place the amplification reaction plate under the condition of -50° C. for 20 minutes to dry, then use a parafilm to seal the amplification reaction plate, and finally place the amplification reaction plate under the condition of 4° C. for use.

[0038] 2. Multiplex tandem PCR

[0039] The first round of PCR amplification reaction system includes: 0.3μL Mix1, 20ng nucleic acid template, 0.2μmol / l external primer mixture (equal amount of each group of external primers added), 25μL Mix2, and finally use ddH 2 O supplemented to 50 μL;

[0040] The amplification process was as follows: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 30 s; annealing at 58°C for 60 s; extension at 72°C for 60 s, a total of 20 cycles; final ex...

Embodiment 3

[0043] Example 3 MT-PCR system specificity verification

[0044]According to the template DNA preparation method, respectively extract the positive recombinant plasmid template DNA containing ITS-1, ITS-2, P0, RACK gene target fragments, and then use the established system to detect the positive control DNA, adenovirus (adenovirus, type 1 and type 4), influenza A viruses (influenza A viruses, H1N1 pdm09, H3N2 and H5N1), influenza B virus (influenza B virus), rhinovirus A (human rhinovirus A) and dengue virus (dengue virus) reverse transcribed DNA to verify the specificity of the system.

[0045] The experimental results show that the system detects positive control DNA, adenovirus, influenza A virus, influenza B virus, rhinovirus A, and dengue virus with a positive coincidence rate and a negative coincidence rate of >95%, which can exclude the interference of other viral nucleic acids , the accuracy is reliable, and the specificity meets the requirements.

[0046] After the ...

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Abstract

The invention discloses a primer, a method and a kit for carrying out multiple tandem PCR (Polymerase Chain Reaction) gene disc detection on leishmania spp. The primer comprises at least two groups of primers, and each group of primers comprises a pair of outer primers and a pair of inner primers. The specific primers are as shown in SEQ ID NO.1 to NO.16. According to the application, the high-throughput detection of the leishmania spp can be simultaneously carried out aiming at the specific DNA fragments of the four types of leishmania spp, the sensitivity is 100%, the specificity is 99.42%, the accuracy can reach 99.55%, the detection time can be shortened, and the detection efficiency can be improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a primer, a method and a kit for performing multiple tandem PCR gene disc detection on Leishmania. Background technique [0002] Leishmaniasis is listed by the World Health Organization as one of the five major parasitic diseases. It is caused by Leishmania spp. It has complex pathogenicity and is widely distributed in Asia, Europe, Africa, and Latin America. . Worldwide, 1.5 to 2 million people are infected with leishmaniasis every year, and more than 80 countries have reported leishmaniasis, posing a threat to 350 million healthy people. The incidence of leishmaniasis in my country is relatively low, maintaining at the level of 0.0372 / 100,000, mainly sporadic cases. Domestic research work on the rapid detection of Leishmania is less, and there is a lack of commercial PCR reagents for rapid detection. Leishmaniasis epidemics continue to emerge in the m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/6893C12Q1/686C12Q2600/16C12Q2600/166C12Q2537/143C12Q2545/113
Inventor 高国龙常晓松何纬邓晓东张青刘杨
Owner 四川国际旅行卫生保健中心(成都海关口岸门诊部)
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