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Primer group for detecting and distinguishing Echinococcus granulosus and Echinococcus multilocularis through loop-mediated isothermal amplification

A ring-mediated isothermal and Echinococcus granulosus technology, applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve problems such as unsatisfactory and incompetent for grass-roots and on-site detection needs, To achieve the effect of strong specificity and high detection sensitivity

Pending Publication Date: 2021-01-15
ZHEJIANG MEDICAL COLLEGE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Polymerase chain reaction (PCR) has extremely high sensitivity and specificity, and has been applied to the diagnosis of various parasitic diseases. However, PCR requires the use of sophisticated instruments and cannot meet the needs of grassroots and on-site detection; The method of real-time quantitative PCR typing of MGB probes to detect Echinococcus multilocularis and Echinococcus granulosus also requires professional experimental technical operators and special detection instruments (fluorescence quantitative PCR instrument), which is not suitable for grass-roots and on-site testing. detection needs

Method used

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  • Primer group for detecting and distinguishing Echinococcus granulosus and Echinococcus multilocularis through loop-mediated isothermal amplification
  • Primer group for detecting and distinguishing Echinococcus granulosus and Echinococcus multilocularis through loop-mediated isothermal amplification
  • Primer group for detecting and distinguishing Echinococcus granulosus and Echinococcus multilocularis through loop-mediated isothermal amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Primer Design

[0036]Primers were designed according to the repeat region sequence (RRS) of Echinococcus granulosus (Gene bank: KR347168.1) and the microsatellite sequence of Echinococcus multilocularis EmTriple83 (NCBI: AF492849.1), providing the isothermal There are 2 sets of 8 primers required for the amplification reaction, including outer primers F3-1, B3-1, F3-2, B3-2, inner primers FIP-1, BIP-1, FIP-2, BIP-2 .

[0037] The first outer primer:

[0038] Outer primer F3-1: 5'-gcatgtgtgtgaatgcaagc-3',

[0039] Outer primer B3-1: 5'-gggcaatcgcagtgaagt-3';

[0040] First pair of internal primers:

[0041] Internal primer FIP-1: 5'-aactacctccacagcacggcagcagatgcctacccatcc-3',

[0042] Internal primer BIP-1: 5'-taagacatcggtgcgagcactctgccttcgttaggtggagat-3';

[0043] The second outer primer:

[0044] Outer primer F3-2: 5'-aaccaccaacctttcggtta-3',

[0045] Outer primer B3-2: 5'-ggaatgggaaggtgatggc-3';

[0046] Second pair of internal primers:

[0047] ...

Embodiment 2

[0049] Example 2: Specific detection of the first outer primer and the first pair of inner primers

[0050] Using the genomic DNA of Echinococcus multilocularis, Echinococcus granulosus, murine tapeworm, Sparganum Guangzhou isolate, Sparganum Henan isolate, Schistosoma japonicum, and Angiostrongylus cantonensis as templates, add each template into the reaction tube and add the primer set F3-1, B3-1, FIP-1, BIP-1 (outer primer final concentration is 5pmol, inner primer final concentration is 40pmol), 8U Bst 2.0 DNA polymerase, 1.5mM dNTP, 7.5mM MgSO 4 and ddH 2 O, in addition, 1 μl of SYTO13 fluorescent dye with a concentration of 25 μM was added, and the reaction was carried out on a fluorescent quantitative PCR instrument of Bio-Rad. The specific reaction procedure was: react at 65°C for 1.5h and collect fluorescence data, and react at 95°C for 5min.

[0051] The result is as figure 1 As shown, the primer set can detect the genomic DNA of Echinococcus granulosus, but cannot...

Embodiment 3

[0052] Embodiment 3: the specificity detection of the second outer primer and the second pair of inner primers

[0053] Using the genomic DNA of Echinococcus multilocularis, Echinococcus granulosus, murine tapeworm, Sparganum Guangzhou isolate, Sparganum Henan isolate, Schistosoma japonicum, and Angiostrongylus cantonensis as templates, add each template into the reaction tube and add the primer set F3-2, B3-2, FIP-2, BIP-2 (outer primer final concentration is 5pmol, inner primer final concentration is 40pmol), 8U Bst 2.0 DNA polymerase, 1.5mM dNTP, 7.5mM MgSO 4 and ddH 2 O, in addition, 1 μl of SYTO13 fluorescent dye with a concentration of 25 μM was added, and the reaction was carried out on a fluorescent quantitative PCR instrument of Bio-Rad. The specific reaction procedure was: react at 65°C for 1.5h and collect fluorescence data, and react at 95°C for 5min.

[0054] The result is as figure 2 As shown, the primer set can detect the genomic DNA of Echinococcus granulosu...

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Abstract

The invention discloses a primer group for detecting and distinguishing Echinococcus granulosus and Echinococcus multilocularis through loop-mediated isothermal amplification. The primer group comprises two pairs of outer primers and two pairs of inner primers, wherein the primers are designed according to an Echinococcus granulosus repeat region sequence and an Echinococcus multilocularis EmTriple83 microsatellite sequence. According to the primer group for detecting and distinguishing Echinococcus granulosus and Echinococcus multilocularis through loop-mediated isothermal amplification, genomic DNA of Echinococcus granulosus or Echinococcus multilocularis contained in a positive sample can be distinguished through two reactions, 1.6 fg of template DNA can be detected to the minimum, thedetection sensitivity is high, and the specificity is high.

Description

technical field [0001] The invention relates to the technical field of molecular detection of parasites, in particular to a primer set for loop-mediated isothermal amplification detection and differentiation of Echinococcus granulosus and Echinococcus multilocularis. Background technique [0002] Echinococcosis is a zoonotic parasitic disease that seriously affects people's health and the development of animal husbandry. It is caused by the larvae of Echinococcus and is a neglected tropical disease. According to the World Health Organization, the number of deaths due to echinococcosis is about 19,300 people worldwide every year, and the disease burden is about 871,000 disability adjusted life years (DALY). In addition, echinococcosis seriously affects the development of animal husbandry, and the global economic loss caused by the disease is as high as 2 billion US dollars every year. The population threatened by echinococcosis in my country is about 50 million, the serologi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6888C12Q1/6844C12Q2531/119C12Q2563/107Y02A50/30
Inventor 孔庆明卓洵辉丁豪杰陆绍红童群波张頲庞华胜杨诗杰艾佳佳罗钊辉
Owner ZHEJIANG MEDICAL COLLEGE
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