Kit and method for detecting human parvovirus B19 by integrating sample treatment, nucleic acid extraction and multiple isothermal amplification

Abstract

The invention provides a kit and a method for detecting human parvovirus B19, which integrate sample treatment, nucleic acid extraction and multiple isothermal amplification. The kit comprises an isothermal amplification detection reagent containing primers of which the nucleotide sequences are SEQ ID NO: 3-10, a reagent for nucleic acid separation / purification, a negative control and a positive control, the isothermal amplification detection reagent and the reagent for nucleic acid separation / purification are converted into freeze-dried powder by adopting a special freeze-drying process. According to the method provided by the invention, the DNA of the sample to be detected is rapidly obtained through the PCR reaction tube containing the micro separation tube, and the human parvovirus B19 is accurately detected by adopting a multiple constant-temperature probe method. According to the kit and the method provided by the invention, one or more defects of the existing isothermal amplification method are overcome, and particularly, 'sample feeding and result discharging 'in a real sense is realized; the detection sensitivity is remarkably improved while the storage and transportation cost is reduced, POCT detection is convenient to realize, and the kit can be widely applied to hospital outpatient emergency treatment and primary medical institutions.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a kit and method for detecting human parvovirus B19 integrating sample processing, nucleic acid extraction and multiple constant temperature amplification. Background technique [0002] Human parvovirus B19 is the smallest known virus, belonging to Parvoviridae, non-enveloped, single-stranded DNA virus with 3500 bases, belonging to Parvoviridae and a species of Erythrovirus, with a diameter of 20-25nm. The resistance to external physical and chemical factors is very strong. The B19 virus can be divided into three genotypes 1, 2, and 3, of which type 1 and type 3 are each divided into two subtypes. [0003] B19 virus infection is ubiquitous and can be transmitted through droplets, skin contact, blood, blood products or vertically through the placenta. The clinical manifestations are different in people of different ages and immune status, and can cause various disea...

AI Technical Summary

Problems solved by technology

B19 nucleic acid detection methods mainly include PCR detection technology and constant temperature detection technology. However, PCR detection technology has the following problems: (1) PCR detection generally relies on repeated operations in multiple stages such as denaturation, annealing and extension, and generally takes a long time. The detection takes 1.5 hours to 2 hours; (2) It is difficult to realize POCT on-site detection, so PCR detection technology has not been promoted and applied at the grassroots level

At this stage, the nucleic acid extraction of samples mainly adopts the spin column method and magnetic bead method. Both methods have to go through steps such as lysis, binding, washing, rinsing, and elution. The operation is complicated, time-consuming, and requires a large amount of equipment. Protein allosteric agents and organic solvents

Sample extraction is likely to cause cross-contamination between samples, resulting in false positives. At the same time, nucleic acid extraction requires professional laboratory equipment and environment, and there are special requirements for operators

[0007] The constant temperature amplification system contains active ingredients such as polymerase and dNTP, which need to be stored at low temperature. When used, it needs to be thawed and repeatedly frozen and thawed. Not only is the operation cumbersome, but it will also affect or even inactivate the active ingredients in the reagent.

In addition, low temperature storage of diagnostic reagents will increase storage, transportation costs and energy consumption

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