Method for extracting viral nucleic acid from animal sample with low nucleic acid content

Abstract

The invention discloses a pretreatment method capable of improving a sample having multiple impurities and complex matrix, such as an animal nasopharyngeal swab and rope biting saliva. The pretreatment method comprises the following steps of: obtaining an animal sample with low nucleic acid content; carrying out protection treatment on the sample with low nucleic acid content; carrying out centrifugal treatment on sample mixed solution, and taking a precipitation part containing the nano magnetic beads at the lower part as to-be-extracted solution for later use; and, carrying out nucleic acidextraction on the to-be-extracted solution by using an automatic nucleic acid extractor, and the like. The problems that a column membrane method is fussy to operate and the nucleic acid recovery rateis not high can be solved; the problems that the sample loading amount of the traditional automatic magnetic bead method is small and virus nucleic acid attached to particles cannot be extracted alsocan be solved; the characteristic that surface modification magnetic beads are convenient to disperse and collect is sufficiently utilized; the operation mode of the traditional automatic magnetic bead method is further optimized; the treatment speed and the nucleic acid recovery rate of the sample having multiple impurities and complex matrix, such as the animal nasopharyngeal swab and rope biting saliva, are greatly increased; a special PCR reagent is matched; therefore, the detection sensitivity can be greatly improved; and generation of false negative can be effectively reduced.

Description

technical field [0001] The invention relates to the technical field of animal disease detection, in particular to a method for improving nucleic acid extraction efficiency before early animal disease detection (fluorescent quantitative PCR method), which is mainly applied to low nucleic acid extraction from animal nasopharyngeal swabs, rope-biting saliva, and the environment. Nucleic acid extraction of content samples. Background technique [0002] In animal disease detection, viral nucleic acid is an important basis for judging whether animals are sick. Therefore, the efficiency of extracting viral nucleic acid from samples directly affects the accuracy of disease detection and judgment. The existing methods for extracting nucleic acid mainly include column membrane method and automatic magnetic bead method. [0003] Among them, the column membrane method is to immobilize the functional groups that can adsorb nucleic acid on the spin column membrane. By adding different ly...

AI Technical Summary

Problems solved by technology

The disadvantage is that the sampling volume is generally only 200 to 300 μL, and it is easy to miss the detection of samples with low virus concentration in the early stage of infection

Another fatal problem is that the viruses in nasopharyngeal swabs and saliva samples from biting ropes of some animals are charged. Because the method cannot handle the impurity particles, it needs to be centrifuged to remove the particles. It is very likely that the viral nucleic acid attached to the particles and impurities will also be removed. When encountering samples with low nucleic acid content, it is easy to lead to extraction failure. Enough target nucleic acid, resulting in false negatives

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