Fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit and detection method of various viruses of donor corneas

Abstract

The invention relates to a fluorescent quantitative PCR detection kit and a detection method of various viruses of donor corneas. The kit comprises an RNA (Ribonucleic Acid) extraction reagent, an RNA reverse transcription and amplification reagent and positive working standards, wherein the RNA reverse transcription (RT) and amplification reagent comprises a 2X RT-PCR premix reagent, polymerases, reverse transcriptases, forward and reverse HCV (hepatitis C virus) primers, an HIV1 (human immunodeficiency virus 1) primer, an RV (rubella virus) primer, HCV, HIV1 and RV fluorescent probes and HCV, HIV1 and RV negative and positive control; the positive working standards are pUC57-HCV plasmids containing destination sequences amplified by the HCV forward and positive primers and pUC57-HIV1 plasmids containing destination sequences amplified by the HIV1 forward and positive primers respectively. 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 detection of donor corneal virus infection by molecular biology techniques, in particular to the rapid detection of severe disease viruses hepatitis C virus (HCV), human immunodeficiency virus type I (HIV1) and rabies virus (RV) in the donor cornea Kits and detection methods. Background technique [0002] The biological safety of eye banks in my country is extremely poor, and the problem of iatrogenic infection of donor corneas has not been controlled. The main reason is that most of the eye bank donors have incomplete information on the general conditions of the donors. Donors who died unexpectedly did not even have medical information on the general conditions, and there is a great risk of infecting various types of hepatitis, rabies and AIDS. In the case of shortage, the clinical use of the donor cornea should be maximized on the premise of ensuring safety. At present, the main problem for eye banks in my coun...

AI Technical Summary

Problems solved by technology

Although the polymerase chain reaction (PCR) 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

Images