Kit for detecting mycoplasma hominis nucleic acid through PCR-fluorescence probe method and detecting method of kit

Abstract

The invention relates to a kit for detecting mycoplasma hominis nucleic acid through a PCR-fluorescence probe method and a detecting method of the kit. The kit comprises a nucleic acid extracting set and a nucleic acid amplification detecting set. The nucleic acid amplification detecting set comprises PCR reaction liquid, negative control and positive control. The PCR reaction liquid comprises DNA polymerase, UNG enzymes, reaction buffer liquid, a primer, a probe and Mg2+. The positive control comprises positive plasmids and human genomes. The negative control comprises human genomes. The probe is a Taqman fluorescence probe. The special primer probe is designed according to an MH polymerase conservative target sequence by means of the kit and the method, the MH is rapidly detected through the PCR-fluorescence probe method, and the result is more reliable and accurate. Compared with an existing culture method, operation is easy and convenient and the speed is fast when mycoplasma hominis is identified; the whole process is completed within 3 hours; specificity and flexibility are high, and the result is visual.

Description

technical field [0001] The invention relates to the field of medical detection, in particular to the field of human virus detection, in particular to a PCR-fluorescent probe method detection kit for Mycoplasma hominis nucleic acid and a detection method thereof. Background technique [0002] According to the national STD epidemic report, 859,040 cases of 8 STDs were reported in 31 provinces in 2003, an increase of 2.59% over the previous year. The reported incidence rate of STDs nationwide is 68.91 / 100,000, and the ratio of male to female cases is 1.4:1. Among the 8 reported STDs in the country, the top 5 are gonorrhea, nongonococcal urethritis (cervicitis), condyloma acuminatum, syphilis and genital herpes. Among them, the number of reported cases of gonorrhea decreased the most compared with the previous year, which was 16.22%. The number of non-gonococcal urethritis (cervicitis) increased the most compared with the previous year, which was 31.71%. Among the 8 types of r...

AI Technical Summary

Problems solved by technology

[0007] Gapdh is used as the target gene detection in the literature of probe detection of MH, but through the database query of GenBank+EMBL+DDBJ+PDB sequences, the conservation of Gapdh of MH is poor, and the primers and probes used in the literature cannot cover all type, and using 2 pairs of primer-probe combinations, each sample requires 2 reactions for detection, which increases the cost and is not simple enough; some use yidC as the target gene detection, but there is a lack of reasonable negative and positive controls

[0008] There are several patents for the detection of Mycoplasma hominis as follows: A kit for cultivating and identifying pathogenic Mycoplasma and its preparation method (CN101109746A), Mycoplasma urogenital tract culture medium and detection method (CN101555457A), Mycoplasma nongonococcal urethritis rapid Detection reagent (CN10156662A), solid medium for rapid detection of mycoplasma and preparation method thereof (CN102409076A), gold label rapid detection kit for mycoplasma hominis (CN104076147A), combined detection kit for chlamydia trachomatis, ureaplasma urealyticum and mycoplasma hominis (CN204142733U ) and compositions and kits (CN101824471A) for the simultaneous detection of Ureaplasma urealyticum, Mycoplasma hominis and Mycoplasma genitalium etc., what the above-mentioned patent part adopted is the culture method to detect Mycoplasma hominis, and the shortcoming is that the culture method identification takes a long time (more than 24 hours), improper material collection or poor culture conditions are prone to false negatives, and the sensitivity is low. The identification requires certain experience in morphological analysis. Due to the long time and many steps in the process from material collection to culture, there are also certain false positives; Some of them use colloidal gold to detect Mycoplasma hominis, which cannot be detected in the early stage of human infection with Mycoplasma hominis. There is a long window period (2-4 weeks), and there are disadvantages such as low sensitivity and insufficient specificity; some use PCR fluorescence The probe method detects Ureaplasma urealyticum, Mycoplasma hominis and Mycoplasma genitalium at the same time, and detects the 16s ribosomal RNA of Mycoplasma hominis. Since the 16s ribosomal RNA of bacteria is evolutionarily conserved, there is a high homology and specificity. low sex, causing false positives

Due to the high detection sensitivity of the PCR fluorescent probe method, there are often some product contamination and non-specific amplification

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