Fluorescence PCR detection reagent capable of discriminating source components of ruminant animal, preparation method and application thereof

Abstract

The invention relates to a biological detection reagent capable of discriminating three types of ruminant animal varieties simultaneously, a preparation method and an application thereof. The invention selects the specificity of the cow, goat and sheep, and the conservative sequence segment of conservative mitochondrial gene as target, applies Primer Eexpress 3.0 software and Primer Select software in DNAStar, and designs and synthesizes a plurality of groups of primers and probes. After being synthesized and marked, the plurality of pairs of designed primers and the probes are carried out best pairing screening experiment so as to obtain the fittest combination of primers and probes. The reagent contains two pairs of specificity primers, and three Taq Man probes, wherein one pair of theprimer aims at cow source components, the other pair of primer aims at common primers of goats and sheep source component detection; the three probes are respective source components to the cows, thegoats and the sheep; and the amplified target fragment length to the source component detection of the cows, the goats and the sheep is respectively 92bp, 136bp and 136bp. The triple Taq Man fluorescence PCR detection method can simultaneously carry out accurate quantitative detection to the source components of the cows, the goats and the sheep, has simple and convenient operation, intuitive result, strong specificity, high sensitivity and good repeatability and can realize high throughput detection of various source components.

Description

technical field [0001] The invention relates to a biological detection reagent capable of simultaneously identifying three ruminant species, as well as a preparation method and application of the reagent. Background technique [0002] In order to prevent the spread of mad cow disease and scrapie through feed, the quarantine department has been testing the ingredients of cattle and sheep origin in imported and exported feed, animal products and cosmetics from mad cow disease and scrapie-affected areas. Purpose. Today, with the rapid development of international trade, it is becoming more difficult to prevent the spread of animal diseases internationally. The adulteration and mixing of different animal-derived products may lead to the accelerated spread of potential animal diseases, and some daily necessities contain different animal diseases. Issues such as the limitation, preference and price difference of origin, such as the fact that halal food cannot contain pig-derived ...

AI Technical Summary

Problems solved by technology

[0008] The above patented method to identify the three ruminant-derived DNAs of cattle, goats and sheep, in addition to the completion of the PCR reaction, an additional melting curve preparation is required, which takes a long time

The addition of fluorescent dyes after PCR may also introduce new contamination factors, resulting in false positive results

In addition, the peak positions of the melting curves exhibited by bovine, goat, and sheep-derived amplicons are relatively close. If there are many interfering substances in the original sample, the three peak shapes are not typical, which may affect the difference between the three ruminants. identification

Images