Fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit and detection method of HBV (Hepatitis B Virus) and TP (Treponema Pallidum) of donor corneas

Abstract

The invention relates to a fluorescent quantitative PCR detection kit and a detection method of HBV and TP of donor corneas. The kit comprises a DNA (deoxyribonucleic acid) extraction reagent, a DNA amplification reagent and positive working standards, wherein the DNA amplification reagent comprises a premix reagent, a 20X fluorescent probe reinforcing agent, forward and reverse HBV primers, an HBV fluorescent probe, forward and reverse TP primers, a TP fluorescent probe, distilled water of ribose-free nuclease and HBV and TP negative control and positive control; the positive working standards are pUC57-HBV plasmids containing destination sequences amplified by the forward and reverse HBV primers and a pUC57-TP plasmids containing destination sequences amplified by the forward and reverse TP primers. The invention realizes the complete closed-tube detection on donor conjunctiva tissue specimens and does not need PCR aftertreatment, and thereby, cross contamination is avoided; meanwhile, real-time detection is adopted, and an obtained Ct value and the original template number are completely in linear correlation; the quantitative accuracy rate is extremely high, and the relative error is about 50 percent, and thereby, the invention is enough to adapt to the demands of clinical nucleic acid quantification.

Description

Technical field

[0001] The invention belongs to the field of molecular biology technology for detecting the infection of donor corneal pathogens, and specifically relates to a rapid detection kit and a detection method for the hepatitis B virus (HBV) and Treponema pallidum (TP), which are the major pathogens of the donor cornea. Background technique

[0002] The biosafety of eye banks in my country is extremely poor, and there is no problem of controlling iatrogenic infections of the donor cornea. Because the data on the whole body condition of the eye bank donor is incomplete, and the accidental death of the donor does not even have the medical data of the whole body condition, there is a great risk of infection of various pathogens, so when the donor cornea is very scarce, it is necessary to On the premise of ensuring safety, try to maximize the clinical use of the donor cornea. At present, the main problem of our country's eye bank is how to establish a set of methods for rap...

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