Fluorescent RT-PCR detection reagent and method for coxsackievirus A16 and enterovirus 71

Abstract

The invention provides a fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) reagent and a method for detecting coxsackievirus A16 and enterovirus 71. A primer and a probe used by the fluorescent RT-PCR detection reagent for the coxsackievirus A16 and the enterovirus 71 provided by the invention are obtained by designing and screening based on the latest gene sequence information of the coxsackievirus A16 and the enterovirus 71 in a public gene sequence database, and have good inclusiveness, specificity and reaction performance; the reagent contains uracil DNA glycosylase and dUTP, so that a false positive result possibly caused by PCR product aerosol can be prevented, and the detection accuracy is further ensured; and besides, the fluorescent RT-PCR detection reagent for the coxsackievirus A16 and the enterovirus 71 is prepared into a freeze-dried reagent which can be stored at normal temperature through a freeze-drying technology, so that the risk that a conventional liquid fluorescent PCR detection reagent fails due to storage temperature change or repeated freeze-thawing is avoided, and the transportation and management cost is reduced.

Description

technical field [0001] The invention relates to pathogenic microorganism detection technology, in particular to the nucleic acid detection of coxsackievirus A16 and enterovirus 71. Background technique [0002] HFMD is a common infectious disease characterized by skin rashes or herpes, fever, sore throat, and general malaise on the hands, feet, and mouth. It was first reported in New Zealand in 1957. Hand, foot and mouth disease is caused by enterovirus infection, mainly through fecal-oral transmission, and can also be transmitted through contact with patient's feces, saliva and herpes fluid. People of different ages can be infected with the disease, mainly children under 5 years old, especially children under 3 years old with the highest incidence rate. [0003] Enteroviruses belong to the genus Enterovirus in the family Picornaviridae. Virus particles are spherical, with a diameter of 28-30 nm. The virus capsid is a three-dimensionally symmetrical icosahedron, composed of...

AI Technical Summary

Problems solved by technology

However, conventional fluorescent RT-PCR detection reagents are all liquid reagents, and there are strict cold chain requirements for transportation and storage (usually -20°C) to maintain reagent activity. will reduce the detection performance of the reagent

Once the traffic is blocked during long-distance transportation, the risk of reagent failure and the cost of cold chain management will increase greatly. It can be seen that liquid reagents cannot be used in long-distance transportation and grass-roots testing institutions with poor cold chain conditions.

[0007] In order to solve the many problems and inconvenience caused by the cold chain requirements of conventional liquid fluorescent RT-PCR detection reagents, a beneficial alternative is to make CA16 and EV71 fluorescent RT-PCR detection reagents into freeze-dried reagents that are stable at room temperature. At present, there is no report of CA16 and EV71 fluorescent freeze-dried RT-PCR detection reagents in the literature

At the same time, due to the increasing number of newly discovered nucleic acid sequences of CA16 and EV71, the fluorescent RT-PCR detection reagents designed based on the original nucleic acid sequence information face an increasing risk of missed detection

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