CN101580867A - Reporting gene amplification kit for detecting mycoplasma - Google Patents
Reporting gene amplification kit for detecting mycoplasma Download PDFInfo
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- CN101580867A CN101580867A CNA200810094566XA CN200810094566A CN101580867A CN 101580867 A CN101580867 A CN 101580867A CN A200810094566X A CNA200810094566X A CN A200810094566XA CN 200810094566 A CN200810094566 A CN 200810094566A CN 101580867 A CN101580867 A CN 101580867A
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Abstract
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:
The invention belongs to biology-molecular nucleic acid detection kit field.
Background technology:
Mycoplasma (Mycoplasma) is the prokaryotic microorganism that a class lacks cell walls, and is very extensive in distributed in nature.From isolating 20 mycoplasma species of human body, 9 kinds of in close relations with human diseases, promptly mycoplasma pneumoniae (M.pneumoniae), Ureaplasma urealyticum (Ureaplasma urealyticum), mycoplasma hominis (M.hominis), mycoplasma genitalium (M.genitalium), mycoplasma fermentans (M.fermentans), penetrate mycoplasma (M.penetrans), Mycoplasma pirum (M.pirum), mycoplasma arthritidis (M.arthritidis) and mycoplasma hyorhinis (M.hyorhinis).Except that to the human body cause illness, mycoplasma is usually infection animal and cell culture also.Since the mycoplasma contamination in reported first cell cultures such as Robinson L B in 1956, of common occurrence about the report of biological products such as mycoplasma contamination cell and vaccine both at home and abroad.Cell cultures (particularly passage cell) is worldwide problem by mycoplasma contamination, cell culture by the mycoplasma contamination rate between 20%～50%.Studies show that both at home and abroad, is following 8 mycoplasma species more than 98%: Mycoplasma orale (M.orale), mycoplasma arginini (M.arginini), mycoplasma hyorhinis (M.hyorhinis), mycoplasma fermentans (M.fermentans), mycoplasma hominis (M.hominis), mycoplasma salivarium (M.salivarium), mycoplasma pulmonis (M.pulmonis) and Lai Shi acholeplasma (A.laidlawii).
Because the acellular wall of mycoplasma can pass through bacterial filter,, already bring a lot of troubles to scientific research and biological products so in cell cultures and production biotechnological formulation process, very easily pollute mycoplasma.Therefore, set up a kind of fast, the mycoplasma method of inspection sensitive, wide spectrum is very necessary.At present, its detection method mainly contains morphological examination (phase microscope, Electronic Speculum), isolated culture, DNA fluorescence colour, serological method (enzyme linked immunosorbent assay, immunofluorescence technique) and nucleic acid probe hybridization method etc., these methods or poor specificity, or sensitivity is low, or cost height, or length consuming time, or complex operation, all can not become the ideal detection means.Along with development of molecular biology, people more and more understand the structure and the molecular composition of mycoplasma.To early 1990s, begun round pcr is applied to the detection of mycoplasma in the world, make detection of mycoplasma become easy, quick, responsive, special, for the detection and the experimental study of mycoplasma have been opened up a wide prospect.European Pharmacopoeia in 2006 with PCR as the conventional sense method.The PCR method that detects mycoplasma at present mainly contains a step PCR (Jia Liyan etc. 2005, Li Mingsheng etc. 2006, Ni Hongxia etc. 2007), nest-type PRC (Guo Yuguang etc. 2005, Dai Yue etc. 2005, Li Yanming etc. 2007), PCR-ELISA (Liu Jin equality 2007), real-time quantitative PCR etc., wherein real-time quantitative PCR is because price factor not popularization and application as yet, and false positive (crossed contamination) and false negative (template is imperfect) result appear in other PCR method easily.The present invention is according to the 16S rRNA gene high conservative region of above-mentioned 14 mycoplasma species, developed a kind of reporter gene amplification kit of detection of mycoplasma, choose one section conserved sequence of mycoplasma as hybridization probe, with one section irrelevant sequence construct reporter gene, with pcr amplification reporter gene again after the sample to be tested hybridization, the shortcoming of evading the easy crossed contamination of conventional PCR.
Reference:
1 Zu Huang that flocks together, Zhao Jiwen pay attention to mycoplasma research and improve 2005 the 7th phase: 677-678 of detection of mycoplasma level China's laboratory medicine magazine
2Robinson?LB，Wichelhausen?RH.Contamination?of?human?cell?cultures?by?pleuropneumonialike?organisms.Science，1956；124：1147-1148.
3 Yin Qiu give birth to, and horse stands the people, the peaceful mycoplasma universal PC of Wang Bei R primer design and 2000 the 3rd phase: the 61-62 of application China's Amphixenosis's magazine that diagnosing
The NESTED-PCR method of using 4 sunlights, Teng Zheng, Zhu Zhaokui detects 2002 the 9th phase: the 414-415 of mycoplasma contamination Shanghai preventive medicine magazine in the cell culture
5 Guo Yu are wide, and Nan Wenlong, Zhang Chunyan etc. use the polymerase chain reaction and detect cell culture and cell cultures 2005 the 7th phase: 30-32 of calf serum mycoplasma contamination China's Animal Quarantine
6 wear happy, Lv Yunwei, detection of mycoplasma and 2005 the 2nd phase: 207-210 of somatotype Peking University journal (medicine) in the clones such as Zhang Chunfeng
7J.Timenetsky，L.M.Santos，M.Buzinhani?et?al.Detection?of?multiple?mycoplasma?infection?in?cell?cultures?byPCR?Braz?J?Med?Biol?Res?39(7)2006：907-914
8V?Gopalkrishna，H?Verma，NS?Kumbhar?et?al?DETECTION?OF?MYCOPLASMA?SPECIES?IN?CELLCULTURE?BY?PCR?AND?RFLP?BASED?METHOD：EFFECT?OF?BM-CYCLIN?TO?CURE?INFECTIONSIndian?Journal?of?Medical?Microbiology，2007?25(4)：364-368
9Hyeran?Sung，Seung?Hye?Kang，Yoon?Jin?Bae?et?al?PCR-Based?Detection?of?Mycoplasma?Species?The?Journalof?Microbiology，2006，Vol.44，No.1：42-49
10 Wu Shang Hui, Peng Cong, cell in vitro such as Gu Huanhua cultivate 2004 the 12nd phase: 111-113 of detection and removing contemporary Chinese medical journal of mycoplasma
11 Lee's open-births, Feng Yuping, the detection of mycoplasma contamination and 2006 the 4th phase: 37-40 of methods analyst Northwest University for nationalities journal (natural science edition) thereof in the serum such as Li Zhuo
12 Ni Hong rosy clouds, Chen Limei, 2007 the 6th phase: 510-512 of detection Beihua University journal (natural science edition) of a thunder mycoplasma species specificity PCR primer design and pair cell culture mycoplasma
13 Jia Li are gorgeous, and Li Yanming opens the foundation of the PCR detection technique of reflecting the live vaccine mycoplasma contamination and uses animal medicine progress, 2005 the 6th phase: 55-58
14 Li Yan are bright, Zhang Ying, and the sleeve type PCR detection technique that Mycoplasma pollutes in the flat live vaccine of Liu Jin is set up and Preliminary Applications China animal doctor magazine, 2007 the 8th phase: 3-5
15 Liu Jin are flat, and Jia Liyan, Jiang Junbing etc. detect the foundation of the PCR-ELISA technology of mycoplasma contamination in the live vaccine and use Journal of Agricultural Biotechnology, 2007 the 1st phase: 102-106
Summary of the invention:
The invention provides " a kind of reporter gene amplification kit of detection of mycoplasma ".
Described invention develops into the reporter gene of mycoplasma base because of haveing nothing to do by probe hybridization crosslinked, the new ideas of the reporter gene indirect detection that increases again with the detection mycoplasma base of routine because of the idea with regard to its gene that increases.It is characterized in that: (lacking sequence with mycoplasma base to be measured because of 16s rRNA complementary is probe with two sections adjacent probes, and two sections sequences are continuous, many bases, just do not form phosphodiester bond between them) to be connected to one section irrelevant reporter gene head and the tail terminal, the reporter gene of band probe can be by head and the tail probe sequence and the complementary hybridization of template to be measured, crossbred strand breach is connected sealing by thermostable ligase near being connected thereby its head and the tail are terminal, makes reporter gene DNA become ring.Adopting reporter gene intermediary sequence again is primer, and oppositely amplification contains the ring-type reporter gene of probe.If there is not specific mycoplasma gene recombination to be measured, reporter gene can not Cheng Huan, also just can not the inverse PCR amplification.Regulate the thermostable ligase reaction conditions, reduce/abandon some superfluous system sensitivities, can make a small amount of crossed contamination insensitive, be difficult for the hybridization reporter gene, also just reduced false positive.If systemic contamination, but with regard to transducer set reporter gene, the remedial measures of many a kind of failures.A described probe sequence length is 10-100base, is preferably 20-30base.Any DNA that described irrelevant reporter gene is and the testing gene sequence is irrelevant, kind and testing gene are far away more good more, and embodiment of the invention employing plant presses down southern mustard LFY conserved sequence as reporter gene.Described reporter gene length is 80-1000base pair (bp), is preferably 200-500bp.Described hybridization-thermostable ligase reaction is 94 ℃ of-50 ℃ of circulations, and cycle number is 1-30, preferred 2-10 circulation.It is 30 ℃-80 ℃ that described thermostable ligase connects temperature, is preferably 50 ℃-60 ℃.Described thermostable ligase is meant Taq ligase.The amplification of described inverse PCR is the short sequence that adopts the reporter gene middle, right half part with the sense strand sequence as upstream inverse PCR primers F, left-half with the antisense strand sequence as downstream inverse PCR primer R, the ring-type that oppositely increases reporter gene.Described test kit composition comprises: band probes report gene, Taq ligase enzyme and damping fluid, 10mM dNTPs, Taq polysaccharase and damping fluid, reverse primer F/R and gel electrophoresis reagent.
Workflow of the present invention is as follows:
1. gene prepares a report: the nucleotide sequence (gg/act/aaactatgtgccagcagc/tcgcggtaatacatagg) of one section high conservative of 16S rRNA gene of selecting 14 mycoplasma species is as hybridization probe.Hybridization probe right half part sequence (sense strand sequence) is added in the synthetic forward primer in one section irrelevant LFY sequence 5 ' terminal sequence front, and left-half sequence (antisense strand sequence) is connected on the synthetic reverse primer in one section irrelevant LFY sequence 3 ' terminal sequence back.Primer with phosphorylation should irrelevant LFY sequence generate reporter gene with the Pfu enzymatic amplification.
2. the hybridization of sample to be tested-reporter gene-ligase enzyme reaction: reporter gene and sample to be measured hybridization, then hybridize if any positive template by its conserved sequence and reporter gene two end sequence, make reporter gene 5 ' end and 3 ' end because of agreeing with in conjunction with template hybridization sequences to be measured is close, the ring-type reporter gene connect to be repaired and generated to its breach through thermostable ligase, and hybridization chain breach both sides base will be mated fully and could be repaired into ring.
3. the inverse PCR of reporter gene: adopt the sequence of LFY middle to carry out inverse PCR according to a conventional method, reflect detected result indirectly as reverse primer.Having used biotin-avidin system simultaneously is the primer mark vitamin H, adds the sepharose particle of the plain mark of an amount of affinity in the electrophoretic buffer, further reduces and pollutes the false positive results that causes.
Advantage:
Hybridization of the present invention-ligase enzyme reactions steps has not only improved system sensitivity, also greatly increased the controllability of system, 1-10 time increase along with heat-resisting ligation cycle index, the detection sensitivity of system will be progressively from the ng level to the pg level and the fg level, the nest-type PRC of maximum sensitivity and secondary TRAP is suitable.Regulate the ratio of heat-resisting ligation cycle number and temperature, reporter gene, also can avoid application of sample to add the crossed contamination of this aerosol glue of reagent markers, be that its positive gene content of real positive sample can effectively detect greater than fg or pg level, and several copy molecules of aerosol glue crossed contamination can not detect between sample.
Description of drawings
Fig. 1 principle of the invention synoptic diagram
Two sections adjacent probes (with template complementary sequence to be measured) of sequence are connected to the terminal formation of the head and the tail reporter gene of one section irrelevant sequence, reporter gene can be by the complementary hybridization of head and the tail and template to be measured, thereby its head and the tail are terminal near being connected, crossbred strand breach is connected enzyme and connects, make reporter gene Cheng Huan, again inverse PCR amplification reporter gene.If there is not the existence of template to be measured, reporter gene can not Cheng Huan, and inverse PCR does not increase.
Fig. 2 the present invention detects the result of mycoplasma in the sample
The 1PCR blank; 2 foetal calf serums 1; 3 foetal calf serums 2; 4 lowlenthal serums; 5E.coli DH5 α;
6 positive controls; 7 reaction solutions of hybridization-connection not; 8 negative controls; The 9DNA standard
Embodiment:
Check the operation that experimentizes of breadboard management regulation in strict accordance with gene amplification: test strict division operation as PCR; Should there be special-purpose gloves, pipettor etc. in each district, must not intersect uses, avoids pollution; The staff should follow folk prescription from a district to two district to the work principle, and each workspace is isolated relatively; Carrying out the working top and the relative article of PCR experiment should regularly sterilize and sterilize with 1% clorox, 75% alcohol, 1mol/L hydrochloric acid or ultraviolet lamp.
1 design of primers
Reporter gene makes up primer: according to the 16S rRNA gene order of 14 mycoplasma species of announcing among the GenBank, with DNAMAN 5.2.2 software analysis, the nucleotide sequence (gg/act/aaactatgtgccagcagc/tcgcggtaatacatagg) of selecting one section high conservative is as hybridization probe.Hybridization probe right half part sequence (sense strand sequence) is added in the synthetic forward primer MyF in one section irrelevant LFY sequence 5 ' terminal sequence front, and left-half sequence (antisense strand sequence) is connected on the synthetic reverse primer MyR in one section irrelevant LFY sequence 3 ' terminal sequence back.MyF：5’-ag?c/t?cgc?ggt?aataca?tag?gtc?gcc?ggt?gac?ac-3’；MyR：5’-gct?ggc?aca?tag?tt?a/t?g?t/c?cca?ctc?tcc?tc-3’
Reporter gene inverse PCR primer: get the long sequence of the about 30bp in reporter gene middle, the sense strand sequence of the about 15base length of right half part is as forward primer RevF, and the antisense strand of the about 15base length of left-half is as reverse primer RevR.RevF5 '-tat gaa gga cga gga g-3 '; RevR:5 '-ccc aca agc gtg ctc-3 ', wherein 5 ' of forward primer RevF end mark vitamin H.
2 genes that prepare a report
Choose structure plasmid pUCId1 behind the sequence synthetic with the irrelevant one section about 300bp of Arabidopis thaliana LFY gene of mycoplasma.Reporter gene with phosphorylation makes up primer and generates reporter gene with this plasmid of Pfu enzymatic amplification.
Plasmid pUCId1 1ul
MyF primer (5uM) 1ul
MyR primer (5uM) 1ul
10×pfu?buffer 5ul
10mM?dNTPs 1ul
pfu?polymerase 1ul
dH
2O 40ul
50ul
The PCR reaction conditions is: 95 ℃ of sex change 3 minutes, 25 circulations, 94 ℃ 35 seconds, 52 ℃ 35 seconds, 72 ℃ 35 seconds.25 the circulation back 72 ℃ 10 minutes.The product Qiagen PCR of company product purification test kit purifying.
The hybridization of 3 samples to be tested and reporter gene-ligase enzyme reaction:
Add sample to be tested 1 μ l, reporter gene 1 μ l, 10 * Taq ligase enzyme damping fluid, 2 μ l, Taq ligase enzyme 0.5 μ l, 15.5 μ l dH in the 20 μ l reaction systems
2O.95 ℃ of sex change 5 minutes, 50 ℃ of incubations 10 minutes.1-2 circulation.
This step adopts ligase chain reaction (Ligase Chain Reaction) thermal cycling to connect catalysis, and the reporter gene ring that first circulation produces can be used as second and takes turns round-robin hybridization template, thereby improves the sensitivity that final PCR detects.
Insert plasmid vector pUC19 after choosing one section mycoplasma nucleotide sequence (about 110bp) synthetic that comprises hybridization probe, make up plasmid pUCMyco as positive control, pUC19 does negative control with plasmid vector.
4 reporter gene inverse PCRs:
Hybridization ligase enzyme reaction solution 2ul
RevF primer (5uM) 1ul
RevR primer (5uM) 1ul
10mM?dNTPs 1ul
10×Taq?buffer 5ul
Taq?polymerase 1ul
dH
2O 39ul
50ul
The PCR reaction conditions is: 94 ℃ of sex change 3 minutes, 25 circulations, 94 ℃ 30 seconds, 52 ℃ 30 seconds, 72 ℃ 30 seconds.Last 72 ℃ 10 minutes.The PCR product adds the sepharose particle of the plain mark of an amount of affinity through the 1.5-2%agarose electrophoretic analysis in the electrophoretic buffer.
5 results
With the present invention testing sample is detected, the PCR blank is with the equivalent ultrapure water of sterilizing, and positive control is plasmid pUCMyco, and negative control is plasmid vector pUC19, and amplification is seen Fig. 2.As shown in Figure 2, positive control produces the specificity product of a treaty 330bp, and blank, negative control does not have the specific amplification band, consistent with theoretical analysis result.4 testing samples are all negative.
Claims (10)
1 " a kind of reporter gene amplification kit of detection of mycoplasma " has nothing to do the conservative 16sRNA gene of mycoplasma by hybridization probe crosslinked reporter gene, reporter gene indirect detection again increases.It is characterized in that default two sections is probe sequence with the short sequence of mycoplasma 16sRNA complementary to be measured, two probe sequences are adjacent, and base is continuous.It is terminal that two sections probes are connected to one section irrelevant reporter gene head and the tail, the reporter gene of band probe can lead to head and the tail probe sequence and the complementary hybridization of template to be measured, thereby its head and the tail are terminal near being connected, crossbred strand breach is connected by thermostable ligase circulation catalysis, make reporter gene DNA become ring, adopting reporter gene intermediary sequence again is primer, and oppositely amplification contains the ring-type reporter gene of probe.If there is not the specificity testing gene to help, reporter gene can not Cheng Huan, also just can not increase by inverse PCR.
2 " a kind of reporter gene amplification kits of detection of mycoplasma " according to claim 1, described pair of probe sequence is and mycoplasma 16sRNA conserved regions complementary dna sequence dna, described pair of probe length is 20-200base, and single probe length is 10-100base.
3 " a kind of reporter gene amplification kits of detection of mycoplasma " according to claim 2, described pair of probe sequence length is 40-60base, single probe length is 20-30base.
4 " a kind of reporter gene amplification kits of detection of mycoplasma " according to claim 1, described reporter gene are and the irrelevant any DNA of mycoplasma gene order that described reporter gene length is 80-1000base pair (bp).
5 " a kind of reporter gene amplification kits of detection of mycoplasma " according to claim 4, described reporter gene length is 200-500bp.
6 " a kind of reporter gene amplification kits of detection of mycoplasma " according to claim 1, described hybridization-thermostable ligase reaction is 94 ℃ of-50 ℃ of circulations, described cycle index is 1-30 time.
7 " a kind of reporter gene amplification kits of detection of mycoplasma " according to claim 6, described hybridization-thermostable ligase reaction cycle number of times is 2-10 time.
8 " a kind of reporter gene amplification kits of detection of mycoplasma " according to claim 6, the connection temperature of described hybridization-thermostable ligase reaction is 30 ℃-80 ℃.
9 " a kind of reporter gene amplification kits of detection of mycoplasma " according to claim 6, the thermostable ligase of described hybridization-thermostable ligase reaction is Taq DNA Ligase.
10 " a kind of reporter gene amplification kits of detection of mycoplasma " according to claim 1, described test kit composition comprises: band probes report gene, Taq ligase enzyme and damping fluid, 10mM dNTPs, Taq polysaccharase and damping fluid, reverse primer F/R and gel electrophoresis reagent.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101831491A (en) * | 2009-03-11 | 2010-09-15 | 北京泰格瑞分子检验有限公司 | System displacement multiple gene magnification technology |
| CN102676658A (en) * | 2012-04-19 | 2012-09-19 | 江苏省农业科学院 | Nested PCR (polymerase chain reaction) detection method for mycoplasma hyorhinis |
| CN101724693B (en) * | 2009-12-24 | 2013-03-13 | 上海吉盛制药技术有限公司 | Primer pair for detecting nested PCR mycoplasmas, detection kit and use method thereof |
| CN105886667A (en) * | 2016-05-17 | 2016-08-24 | 河南牧业经济学院 | Detection kit for porcine epidemic diarrhea virus and detection method thereof |
| CN109207637A (en) * | 2018-09-30 | 2019-01-15 | 河南牧业经济学院 | A kind of replaceable Ankara kit for detecting nucleic acid of reporting system and detection method |
| CN109251963A (en) * | 2018-11-12 | 2019-01-22 | 复旦大学 | The method and kit of mycoplasma contamination in Constant Temperature Detection cell culture fluid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831491A (en) * | 2009-03-11 | 2010-09-15 | 北京泰格瑞分子检验有限公司 | System displacement multiple gene magnification technology |
| CN101724693B (en) * | 2009-12-24 | 2013-03-13 | 上海吉盛制药技术有限公司 | Primer pair for detecting nested PCR mycoplasmas, detection kit and use method thereof |
| CN102676658A (en) * | 2012-04-19 | 2012-09-19 | 江苏省农业科学院 | Nested PCR (polymerase chain reaction) detection method for mycoplasma hyorhinis |
| CN102676658B (en) * | 2012-04-19 | 2014-06-04 | 江苏省农业科学院 | Nested PCR (polymerase chain reaction) detection method for mycoplasma hyorhinis |
| CN105886667A (en) * | 2016-05-17 | 2016-08-24 | 河南牧业经济学院 | Detection kit for porcine epidemic diarrhea virus and detection method thereof |
| CN109207637A (en) * | 2018-09-30 | 2019-01-15 | 河南牧业经济学院 | A kind of replaceable Ankara kit for detecting nucleic acid of reporting system and detection method |
| CN109251963A (en) * | 2018-11-12 | 2019-01-22 | 复旦大学 | The method and kit of mycoplasma contamination in Constant Temperature Detection cell culture fluid |
| CN109251963B (en) * | 2018-11-12 | 2022-11-18 | 复旦大学 | Method and kit for detecting mycoplasma pollution in cell culture solution at constant temperature |
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