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One-tube nested real-time quantitative PCR detection kit for micro-sample typing and detection of Echinococcus multilocularis and Echinococcus granulosus

A technology for Echinococcus multilocularis and Echinococcus granulosus, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve low copy number, aerosol pollution, nested PCR Difficult clinical diagnosis and other problems, to achieve the effect of high sensitivity, high accuracy, and easy operation

Active Publication Date: 2021-05-14
QINGHAI UNIVERSITY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Regarding the second problem, although the primers and probes of the previously applied invention patent (application number: 201611198588.1) happen to be located within this sequence, the primers and probes of the prior patent cannot be detected from the patient's body due to reasons such as low copy number. Echinococcus nucleic acid information, in view of this, the inventors further considered to improve the detection limit of the original method by designing external primers for nested PCR. The external primers were pre-amplified to increase the amount of the target sequence in the template and then the The first pre-amplification product was used as a template for the second round of amplification
However, the two-step operation of nested PCR needs to open the cover and add samples, which will cause the risk of aerosol contamination, which is why nested PCR is difficult to use in clinical diagnosis.

Method used

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  • One-tube nested real-time quantitative PCR detection kit for micro-sample typing and detection of Echinococcus multilocularis and Echinococcus granulosus
  • One-tube nested real-time quantitative PCR detection kit for micro-sample typing and detection of Echinococcus multilocularis and Echinococcus granulosus
  • One-tube nested real-time quantitative PCR detection kit for micro-sample typing and detection of Echinococcus multilocularis and Echinococcus granulosus

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Embodiment Construction

[0037] The technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Of course, the described embodiments are only some, not all, embodiments of the present invention, and do not imply any limitation to the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0038] In this example, the present invention finds that CBLO020001206 can be used for the in vitro diagnosis of Echinococcus multilocularis and Echinococcus granulosus infection based on the sequencing of free DNA in the plasma of echinococcosis patients, and designs and screens out Echinococcus multilocularis and Echinococcus granulosus infection The outer primer pair, inner primer pair, inner quality control gene primer pair and probe were designed for the specific...

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Abstract

The invention discloses a one-tube nested real-time quantitative PCR detection kit for typing and detecting Echinococcus multilocularis and Echinococcus granulosus in trace samples, and the selection of internal and external primers, probes and PCR amplification conditions is designed The one-tube method of nested PCR is achieved, and the aerosol pollution caused by opening the cover is avoided during the operation, which improves the accuracy of the test results. The detection kit contains PCR internal and external primer pair sequences, MGB typing detection probe sequence, human-source internal reference gene primer pair sequence and probe sequence for internal quality control. The present invention can detect samples containing Echinococcus multilocularis and / or Echinococcus granulosus according to the typing of trace samples such as patient serum circulating DNA, and has high accuracy, and can accurately type and detect Echinococcus multilocularis and Echinococcus granulosus. Specific DNA sequence of Echinococcus granulosus; it can quickly type and identify Echinococcus multilocularis and Echinococcus granulosus in large quantities; it is easy to operate, and can amplify trace DNA in samples and reach the level of typing detection .

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a real-time quantitative PCR detection kit for typing and detecting Echinococcus multilocularis and Echinococcus granulosus. Background technique [0002] Echinococcosis (Echinococcosis) is a zoonotic parasitic disease, also known as echinococcosis, mainly caused by Echinococcus granulosus in my country: unilocular / granular echinococcosis (Cystic Echinococcosis, CE) and larval disease caused by Echinococcus multilocularis: multilocular echinococcosis (Alveolar Echinocococosis, AE). Echinococcosis infection focus grows slowly in the human body, and the clinical manifestations are complicated, and there are no obvious symptoms and signs for a long time. At present, there is still a lack of effective and sensitive methods for the diagnosis and classification of echinococcosis, mainly relying on imaging diagnosis (B-ultrasound, MRI, CT), and often patients diagnosed by i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12Q1/6888C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/16C12Q2600/166C12Q2531/113C12Q2547/101C12Q2549/119C12Q2545/101C12Q2545/113C12Q2563/107C12Q2537/143
Inventor 汤锋格日力樊海宁李润乐赵连娣郑佳张玉英冯琳
Owner QINGHAI UNIVERSITY
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