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

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...

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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.

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