One-tube nested real-time quantitative PCR detection test kit for detecting alveolar echinocococosis and cystic echinococcosis by micro-sample typing

Abstract

The invention discloses a one-tube nested real-time quantitative PCR detection test kit for detecting alveolar echinocococosis and cystic echinococcosis by micro-sample typing. The test kit is characterized in that inner and outer primers, probes and selection of PCR amplification conditions are designed to finish nested PCR by a one-tube method, and aerosol pollution caused by uncovering is avoided in the operation process; and the detection result accuracy is increased. The detection test kit contains PCR inner and outer primer pair sequences, an MGB typing detection probe sequence, a humanized internal reference gene primer pair sequence for internal quality control and a probe sequence. According to the test kit, samples containing alveolar echinocococosis and / or cystic echinococcosiscan be detected by typing aiming at trace samples such as serum circulating DNA of a patient, the accuracy is high, and alveolar echinocococosis and cystic echinococcosis source specific DNA sequencescan be accurately detected by typing; the alveolar echinocococosis and cystic echinococcosis can be quickly classified and identified on a large scale; and the operation is simple and convenient, andthe trace insect source DNA in the sample can be amplified to reach the typing detection level.

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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