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...

AI Technical Summary

Problems solved by technology

The serum immunology method can be completed by the laboratory. This method is cheap and easy to operate, but has low sensitivity and is prone to false negatives. It can be used as a primary screening method

Moreover, this method cannot be implemented when donor blood samples cannot be obtained; genetic testing can be divided into qualitative testing and quantitative testing, and the current research development trend is the rapid development of new technologies that can be quickly and quantitatively tested with low cost and easy to promote

Although the polymerase chain reaction technology has been widely used in nucleic acid analysis, the false positive pollution and quantitative accuracy of ordinary PCR have always troubled people.

It relies on the post-processing of various types of PCR, and these processing processes can easily cause a large number of PCR products to fly into the air, making the false positive contamination of PCR the biggest problem in large-scale clinical applications

On the other hand, the quantification of all these methods is carried out for the final product of PCR, and the platform effect of PCR greatly interferes with the correlation between the number of original templates of PCR and the final product, making it difficult to improve the quantitative accuracy

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