Amplification primer for detecting echinococcosis through ddPCR and construction method and application of amplification primer

Abstract

The invention discloses a primer construction method for detecting hepatic echinococcosis through ddPCR, an amplification primer and application of the amplification primer. The primer construction comprises the following steps that common gene segments of echinococcus granulosus and echinococcus multilocularis are screened, and a plurality of first primers are designed based on the common gene segments; a plasma cfDNA sample of a first patient group is amplified by adopting the first primers, and the first primers capable of amplifying the plasma cfDNA sample of the first patient group are taken as second primers; and a plasma cfDNA sample of a second patient group is detected by adopting ddPCR of a reaction system comprising the second primers, and the second primers with the positive detection rate greater than a preset value are screened out as a target amplification primer. The amplification primer can amplify DNA fragments of echinococcus which are released into peripheral blood and exist in the form of plasma free DNA, by combining with a ddPCR method, whether human plasma is infected with echinococcosis can be diagnosed by detecting the human plasma, so that the ultra-early diagnosis of echinococcus granulosus and echinococcus multilocularis is realized, and a detection tool which is sensitive enough is provided for research and development of an echinococcosis precise therapy.

Description

technical field [0001] The invention relates to the field of echinococcosis diagnosis, in particular to a method for constructing amplification primers for detecting hepatic echinococcosis in peripheral blood based on microdroplet digital PCR, and the amplification primers screened by the method and applications thereof. Background technique [0002] Hydatid disease, also known as echinococcosis, is a parasitic disease caused by echinococcus larvae parasitizing in humans or animals. The infection of echinococcosis can cause damage to organs and tissues such as liver, lung, brain and bones, and the patient loses the ability to work. [0003] Echinococcosis in China mainly includes cystic echinococcosis (CE) and alveolar echinococcosis (AE). Echinococcus larvae parasitize human tissues and organs, while alveolar echinococcosis is caused by alveolar echinococcosis larvae of Echinococcosis multilocularis (Em). [0004] In order to achieve the preventable, controllable and cura...

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Problems solved by technology

[0007] An object of the present invention is to provide a method for constructing amplification primers for detecting hepatic echinococcosis based on droplet digital PCR (ddPCR), the amplification primers obtained by the construction method can be amplified and released into peripheral blood Plasma free DNA (cell-free DNA, cfDNA) in the plasma, and then use ddPCR to detect the copy number in the plasma sample to determine whether the sample is positive, which solves the problem that early diagnosis cannot be realized due to the detection of lesion tissue in the prior art problem, realized the ultra-early diagnosis of Echinococcus granulosus and Echinococcus multilocularis, which can effectively shorten the period of judging the effect of drug treatment, and provide a sensitive enough detection tool for the development of precise therapy for echinococcosis

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