Reporting gene amplification kit for detecting mycoplasma
A technology of reporter gene and mycoplasma, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve the problems of easy cross-contamination, incompleteness, false positives, etc. of PCR
Inactive Publication Date: 2009-11-18
BEIJING TAG ARRAY MOLECULAR TEST +2
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At present, the PCR methods for detecting mycoplasma mainly include one-step PCR (Jia Liyan et al. 2005, Li Mingsheng et al. 2006, Ni Hongxia et al. 2007), nested PCR (Guo Yuguang et al. 2005, Dai Yue et al. ELISA (Liu Jinping 2007), real-time quantitative PCR, etc., among which real-time quantitative PCR has not yet been widely used due to price factors, while other PCR methods are prone to false positive (cross-contamination) and false negative (template incomplete) results
According to the highly conserved region of the 16S rRNA gene of the above 14 kinds of mycoplasma, the present invention has developed a reporter gene amplification kit for mycoplasma detection, selects a conservative sequence of mycoplasma as a hybridization probe, constructs a reporter gene with an unrelated sequence, and After the sample is hybridized, the reporter gene is amplified by PCR, which avoids the disadvantage of easy cross-contamination of conventional PCR
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[0033] Strictly follow the management specifications of the gene amplification testing laboratory to conduct experimental operations: such as PCR experiments are strictly divided into zones; each zone should have special gloves, pipettes, etc., and cross-use is not allowed to avoid contamination; The principle of one-way work in the area, each work area is relatively isolated; the work table and related items for PCR experiments should be sterilized and disinfected regularly with 1% sodium hypochlorite, 75% alcohol, 1mol / L hydrochloric acid or ultraviolet light.
[0034] 1 Primer design
[0035] Reporter gene construction primers: According to the 16S rRNA gene sequences of 14 species of mycoplasma published in GenBank, analyze with DNAMAN 5.2.2 software, and select a highly conserved nucleic acid sequence (gg / act / aaactatgtgccagcagc / tcgcggtaatacatagg) as a hybridization probe. Add the right half of the hybridization probe sequence (sense strand sequence) to the 5' end of an ir...
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The difficult problem of the contamination of cell cultured mycoplasma and the infection diagnosis of various mycoplasma urgently need a reliable and effective kit. Mycoplasma culture by a fluorescent staining method is reliable but takes time and trouble, and culture pollution is intensified. A direct PRC method for amplifying a mycoplasma gene, which is used for the routine detection of the mycoplasma is easy for cross contamination, and high false masculinity is only used as an auxiliary reference. The invention relates to a mycoplasma conservative gene which across links a plant arabidopsis LEY sequence into a reporting gene DNA by a hybridization probe and amplifies the indirect detection of a reporting gene. The mycoplasma conservative gene is characterized in that mycoplasma 16sRNA is hybridized with the head part and the tail part of the LEY reporting gene DNA whose head part and tail part are provided with mycoplasma conservative sequence probes; the reporting gene DNA is catalyzed into a ring by heat-proof ligase, and the annular reporting gene is reversely amplified by PCR so as to indirectly reflect the detection of the mycoplasma; by adjusting ligase reaction, the reporting gene is difficultly across linked by the cross contamination, thereby reducing the false masculinity; and if a system is contaminated, the reporting gene can be changed.
Description
Technical field: [0001] The invention belongs to the field of biology-molecular nucleic acid detection kits. Background technique: [0002] Mycoplasma (Mycoplasma) is a kind of prokaryotic microorganism lacking cell wall, which is widely distributed in nature. Among the 20 species of mycoplasma isolated from the human body, 9 species are closely related to human diseases, namely, mycoplasma pneumoniae (M. pneumoniae), ureaplasma urealyticum (Ureaplasma urealyticum), mycoplasma hominis (M. ), M. fermentans, M. penetrans, M. pirum, M. arthritidis and M. hyorhinis. In addition to being pathogenic to humans, mycoplasma often infects animals and cell cultures. Since Robinson L B et al first reported mycoplasma contamination in cell culture in 1956, there have been frequent reports of mycoplasma contamination of cells and vaccines and other biological products at home and abroad. It is a worldwide problem that cell culture (especially subcultured cells) is contaminated by mycop...
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Inventor 张辉江洪
Owner BEIJING TAG ARRAY MOLECULAR TEST
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