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Primer and kit for simultaneously detecting two types of echinococcosis

A kit and echinococcosis technology, applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems that cannot meet the needs of rapid diagnosis and large-scale investigation, inapplicable control work, and method processes cumbersome and other problems, to achieve the effect of overcoming the lack of accuracy, shortening the experiment time, and high sensitivity

Active Publication Date: 2019-05-17
四川省疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing PCR detection method for Echinococcus is mainly based on single-tube amplification. A pair of primers can only detect the corresponding one Echinococcus through a single reaction, or use sequencing to identify it. In areas with mixed epidemics, the method is cumbersome, time-consuming and consumable, and cannot meet the needs of rapid diagnosis and large-scale investigation
According to public reports and data, there are currently no multiplex PCR primer pairs that can be used to simultaneously detect fine-grained, multilocular, and canadensis. Prevention and control of Echinococcus tapeworm in my country

Method used

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  • Primer and kit for simultaneously detecting two types of echinococcosis
  • Primer and kit for simultaneously detecting two types of echinococcosis
  • Primer and kit for simultaneously detecting two types of echinococcosis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The design of embodiment one primer

[0023] Search and download the mitochondrial gene references of Echinococcus granulosus narrow sense (NC_008075.1), Echinococcus canadensis (NC_011121.1, AB235848.1, MG597240.1) and Echinococcus multilocularis (NC_000928.2) published on GenBank For the sequence, primer Premier 6.0, Oligo 7 software and artificial methods were used to design specific primers for the genes of the above three insect species, and the quality of the primers was evaluated. After multiple rounds of screening, it was finally determined that 3 pairs of primers can quickly and accurately detect and identify 3 species of Echinococcus simultaneously. The primer sequences are:

[0024] (1) Echinococcus granulosus in the narrow sense

[0025] Upstream primer F1: 5'-gtctgtgtttcttaccattg-3' (SEQ ID NO.1)

[0026] Downstream primer R1: 5'-gacccgtacaaacatatatcaac-3' (SEQ ID NO.2)

[0027] (2) Echinococcus canadensis

[0028] Upstream primer F2: 5'-gtaagtctaagttgg...

Embodiment 2

[0033] The establishment of embodiment two multiplex PCR amplification method

[0034] 1. Extraction of genomic DNA from the sample to be tested

[0035] Take 15-25 mg of parasite tissue or human or animal infection focus, and extract the whole genome DNA of the sample according to the instructions of the tissue DNA extraction kit.

[0036] 2. Amplification system

[0037] Use 25 μl reaction system, add 12.5 μl of 2×Taq PCR amplification reagent, 0.5 μl of upstream and downstream primers of 10uM F1 / R1 and F2 / R2 primer pair, 1.5 μl of upstream and downstream primers of 10uM F3 / R3 primer pair, nuclease-free water 6.5μl, sample template DNA 1μl. Using nuclease-free water as a template, the system was prepared according to the above method as a negative control.

[0038] 3. Reaction conditions

[0039]Put the above-prepared PCR reaction tube into the PCR machine, and set the reaction program according to the following conditions: pre-denaturation at 94°C for 2 minutes; denatur...

Embodiment 3

[0040] Identification of embodiment three PCR reaction products

[0041] Take 10 μl of the PCR amplification product and electrophoresis at a constant voltage of 3 to 5 V / cm in 2% (w / v) agarose gel for 30 to 60 min, observe the results in the gel imaging analysis system, and use the DNA molecular weight standard as a reference, According to the presence and size of the target band, determine whether there is infection and the species of Echinococcus infection.

[0042] If the length of the amplified product band is 811bp, it is judged as positive for Echinococcus granulosus infection in the narrow sense; if the length of the amplified product band is 315bp, it is judged as positive for Echinococcus canadensis infection; 457bp, it is judged to be positive for Echinococcus multilocularis infection; if the amplified product has the above two or three length bands at the same time, it is judged to be positive for the mixed infection of the corresponding two or three kinds of Echin...

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Abstract

The invention discloses a primer and a kit for simultaneously detecting two types of echinococcosis. The primer is arranged in the kit in the form of freeze-dried powder or dissolved in a buffer solution. The primer comprises three pairs of specific primer pairs respectively comprising: an echinococcus granulosus specific primer pair, an echinococcus canadensis specific primer pair and an echinococcus multilocularis specific primer pair. By utilizing the three pairs of primers in the kit, individual or mixed infection of the three pathogens can be detected by one round of PRC amplification, and it can be determined whether samples to be detected are infected and infected with the species or not according to the specific bands present in the electrophoresis of the amplification products. Byadopting the kit, not only is the operation fast and convenient, but also the hydatidosis caused by three echinococcus infections can be accurately diagnosed and distinguished.

Description

technical field [0001] The invention relates to a primer and a kit, in particular to a primer and a kit for simultaneously detecting two types of echinococcosis. Background technique [0002] Hydatid disease is caused by the infection of multiple species of Echinococcus, which seriously endangers human health and causes a huge social and economic burden. Echinococcosis is mainly divided into two types: cystic and vesicular. The former is widely distributed, and the global disease burden of patients reaches 10,096.62 million DALYs (disability-adjusted life years), causing direct economic losses of more than 3 billion yuan in my country every year; the latter Also known as "worm cancer", it can lead to liver cancer-like lesions and has a very high fatality rate. If left untreated for ten years, the mortality rate can reach 94%. China is one of the countries where echinococcosis is most prevalent in the world. The estimated number of people suffering from echinococcosis has rea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCY02A50/30
Inventor 尚婧晔黄燕张光葭钟波王谦喻文杰何伟廖沙王奇李调英陈兴旺
Owner 四川省疾病预防控制中心
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