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 detection of pathogenic infection of donor cornea, and particularly relates to a rapid detection kit and detection method for hepatitis B virus (HBV) and Treponema pallidum (TP), major pathogenic pathogens of donor cornea. Background technique [0002] The biosafety of eye banks in my country is extremely poor, and the problem of iatrogenic infection of the donor cornea is not controlled. Because the information on the general condition of eye bank donors is incomplete, the donor who died unexpectedly does not even have medical information on the general condition, and there is a great risk of infection with various pathogens. Therefore, when the donor cornea is very scarce, it is necessary to Under the premise of ensuring safety, the scope of clinical use of the donor cornea should be maximized. At present, the main problem of my country's eye bank is how to establish a method to use ...

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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