CN102787162A - Kit for detection of abortion chlamydia psittaci and method - Google Patents
Kit for detection of abortion chlamydia psittaci and method Download PDFInfo
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- CN102787162A CN102787162A CN2011101431862A CN201110143186A CN102787162A CN 102787162 A CN102787162 A CN 102787162A CN 2011101431862 A CN2011101431862 A CN 2011101431862A CN 201110143186 A CN201110143186 A CN 201110143186A CN 102787162 A CN102787162 A CN 102787162A
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Abstract
The present invention relates to a kit for detecting microorganisms, particularly a kit for detection of abortion chlamydia psittaci for pigs, sheep and cattle detection, and a preparation method and a use method thereof. The kit for detection of abortion chlamydia psittaci of the present invention comprises two pairs of specific primers composed of an outer primer and an inner primer, wherein the sequence of an upstream outer primer F3 is SEQ ID No. 1; the sequence of a downstream outer primer B3 is SEQ ID No. 2; the sequence of an upstream inner primer FIP is SEQ ID No. 3; and the sequence of an downstream inner primer BIP is SEQ ID No. 4. The kit of the invention has high sensitivity and high specificity, is simple and convenient in the detection method, and can be applicable to detection of pigs, sheep and cattle.
Description
Technical field
The present invention relates to a kind of test kit that detects mikrobe, particularly a kind of test kit that pig, sheep and Niu Jinhang abortus Chlamydia psittaci are detected with and preparation and method of use.
Background technology
Chlamydozoan (Chiamydiaceae) is one type of entozoic gram-negative micro-organism of special sexual cell between bacterium and virus, and size is 0.2～0.4 μ m.Chlamydia psittaci (C.psittaci) is as a crucial kind in the chlamydiaceae, is the sick main pathogenic bacterium of animal chlamydia, has host widely, in the domestic animal with ox, sheep, pig susceptible comparatively.Can cause by fetus die young, significantly minimizing and feed, manpower waste and the tremendous economic loss that causes has caused great harm to aquaculture of milk yield.In addition, all right infected person of abortus Chlamydia psittaci threatens human health.
The sick research of animal chlamydia started from for 19 end of the centurys; Marange (1892) finds in Buenos Aires, Argentinian capital; The people who contacts with the parrot bird understands sudden onset; Thereby finally affirmed the parrot bird the human infection with fall ill in vital role, and this new name of disease of psittacosis (Psittacosis) proposed.Present this disease most countries all over the world and area.Chlamydozoan can cause the salpingitis of mammiferous miscarriage, pneumonia, enteritis, encephalomyelitis, polyserositis, polyarthritis, keratoconjunctivitis, urethritis and chicken etc.According to the livestock and poultry epidemiology survey of suburb, Beijing is found that some plants have all found the chlamydozoan cause of disease in various degree, have caused property miscarriage of boar chlamydia psittaci and pneumonia, even in the kind goat of import, also have chlamydia psittaci.Calendar year 2001, the Lanzhou veterinary institute detects 343 parts of buffalo serum I HAT that pick up from Guangxi, 12 counties in Anhui and Jiangsu, finds that all there is choamydiae infection in various degree in each county buffalo crowd, and positive rate is minimum to be 3.3%, is up to 39.3%.Because the sick initial infection multilist of animal chlamydia is existing not obvious and other many illnesss have similarity, therefore is easy to obscure.
China cultivates the main applying biological of the sick detection method of animal chlamydia and serological test (" livestock and poultry chlamydiosis and control thereof ", Qiu Changqing chief editor) at present.Though biological culture is accurately and reliably, length consuming time, complicated operation can't detect on a large scale; Though IHA and immune chromatography test paper method are simple and easy to operate in the serological method; But equally with the ELISA detection method all can't differentiate animal chlamydia natural infection or immunological status; Also exist certain cross reaction and personal errors hidden danger; In addition, ELISA detects special instruments such as also needing ELIASA.These have brought very big difficulty all for clinical quarantine.Iodine and Giemsa staining also are that this disease detects one of method commonly used, and referring to " livestock and poultry chlamydiosis and control thereof " (Qiu Changqing chief editor), but this method is not suitable for directly being used for the detection of clinical sample.Along with development of molecular biology, regular-PCR method (Zhou Jizhang etc., 2007) because of its sensitivity, fast, need not advantage such as viable bacteria operation, be used for this cause of disease in a large number to detect, but needed special instrument, complex operation, be not suitable for clinical and basic unit produces use.Rely on nucleotide sequence amplification technique (NASBA) though easier, quick, special, and be used for the detection of multiple virus, use NASBA and in detection, have problems (high lightning etc., 2009) such as insufficient sensitivity and false positive some cause of diseases.Therefore, the zoonosis that causes along with chlamydia psittaci is on the rise, and abortus Chlamydia psittaci detection methods such as the ox that efficient, quick, the accurate and suitable basic unit of foundation produces, sheep, pig are more and more urgent.
Summary of the invention
The present invention provides a kind of prior art deficiency that overcomes, and is convenient to use in basic unit, detects weak point consuming time, and cost is low, the test kit of detection ox simple to operation, sheep, pig abortus Chlamydia psittaci, and the preparation method of this test kit and method of use.
Include two pairs of Auele Specific Primers being made up of outer primer and inner primer in the test kit of detection abortus Chlamydia psittaci of the present invention, wherein: the sequence of upper reaches outer primer F3 is SEQ ID № 1; The sequence of downstream outer primer B3 is SEQ ID № 2; The sequence of upper reaches inner primer FIP is SEQ ID № 3; The sequence of downstream inner primer BIP is SEQ ID № 4.
Use for convenient, also can place trimethyl-glycine, sal epsom, dNTPs, ThermoPol reaction buffer and Bst archaeal dna polymerase in the test kit; Perhaps also be added with developer SYBR GREEN I.
The method of use of the test kit of detection abortus Chlamydia psittaci of the present invention is:
A. be mixed with the LAMP reaction solution with two pairs of special primers in the test kit;
B. from test sample, extract DNA;
C. be that template and LAMP reaction solution are mixed with the LAMP reaction mixture with DNA;
D. add the Bst archaeal dna polymerase at the LAMP reaction mixture and carry out the LAMP reaction;
Carry out agarose electrophoresis behind the e.LAMP reaction terminating, according to presenting the positive and negative of the test sample of gradient band judgement clearly behind the reaction product electrophoresis; Perhaps add the direct visual inspection of developer SYBR GREEN I,, contain abortus Chlamydia psittaci in the decidable sample if reaction solution presents green; If it is orange that reaction solution appears, then judge not contain abortus Chlamydia psittaci in the sample.
Susceptibility and specificity to test kit among the present invention are verified.Agarose electrophoresis result shows that the susceptibility that test kit detects C.psittaci is identical with conventional sleeve type PCR method, and detection limit is 4 copies; The test kit specificity is high; And all negative to the detected result of chlamydia trachomatis, CPN, Brucella, intestinal bacteria, vibrio cholerae and clostridium perfringens when using detection kit of the present invention to detect, can not influence the result of detection because of the existence of these mikrobes.
Can be known that by foregoing the present invention utilizes loop-mediated isothermal amplification to detect abortus Chlamydia psittaci to infect, its advantage is: the test kit among (1) the present invention has hypersensitivity; (2) test kit among the present invention has high specific; (3) do not need thermal cycle reaction, needn't in the PCR appearance, increase, only need simple thermostat, get final product like water-bath etc.; (4) compare with the conventional PCR of 2-5h consuming time, the reaction times shortens greatly, generally only needs 1-2 hour; (5) its detected result can be according to the colour-change naked eyes result of determination of reaction solution, and is simple and convenient; (6) can make quick diagnosis usefulness in small test chamber and plant of basic unit; (7) the present invention is applicable to pig, sheep and Niu Jinhang are detected.
Description of drawings
Fig. 1 is to use developer to carry out result's judgement.Be followed successively by positive control, positive, negative control and blank pipe from left to right.
Fig. 2 is the susceptibility comparing result that test kit and sleeve type PCR method utilization agarose electrophoresis detect C.psittaci.Swimming lane M is DL2000Marker; Swimming lane P is a positive control; Swimming lane N is a negative control; Swimming lane 1-9 (A) detects 4 * 10 respectively with test kit
7, 4 * 10
6, 4 * 10
5, 4 * 10
4, 4 * 10
3, the electrophoresis result of the C.psittaci SX5 strain dna profiling of 400,40,4,0.4 copies, this method can detect the dna profiling of 4 copies; Swimming lane 1-9 (B) detects 4 * 10 respectively with the sleeve type PCR method
7, 4 * 10
6, 4 * 10
5, 4 * 10
4, 4 * 10
3, the electrophoresis result of 400,40,4,0.4 copy C.psittaci SX5 strain dna profilings, this method also can detect the dna profiling of 4 copies.
Embodiment
Below in conjunction with embodiment the present invention is further described, but the present invention is not limited to the content of embodiment.
(1) preparation of test sample
The C.psittaci vaccine strain D13 of abortus Chlamydia psittaci, A and SX5 are separated to from pig, the Yang Heniu that the miscarriage symptom is arranged respectively, and through the animal doctor of the Ministry of Agriculture of Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences public health emphasis open laboratory's isolation identification and preservation; The plasmid pMD-MOMP that contains SX5 strain MOMP gene is made up by this laboratory; The plasmid Cpn0695-pGEX-6p that contains CPN (C.pneumoniae) MOMP gene is so kind as to give by health science center immunity of texas,U.S university and the bright professor of department of microbiology clock; Chlamydia trachomatis, Brucella, intestinal bacteria, vibrio cholerae and clostridium perfringens bacterial strain are preserved by this laboratory.
(2) set up the LAMP test kit that detects abortus Chlamydia psittaci, this test kit can detect 50 duplicate samples.
1, two couples of special primer F3, B3, FIP and BIP are provided.Wherein F3 is a upper reaches outer primer, and B3 is the downstream outer primer, and FIP is a upper reaches inner primer, and BIP is the downstream inner primer.Primer sequence is following:
Upper reaches outer primer F3:5 '-ACCTCTAACAGCTGGTACTG-3 '
Downstream outer primer B3:5 '-TGGGTTCCATGTGGTCAAG-3 '
Upper reaches inner primer FIP:
5’-GAGAGCGCTAAACCAACTTGCCAACTGACACTAAGTCGGCT-3’
Downstream inner primer BIP:
5’-GGCATAAACTGGTCACGAGCAATTTAGGTTGAGCGATGCGG-3’
2, also comprise following reagent in the test kit
The LAMP reaction solution of 22 μ l is housed in the centrifuge tube of (1) 50 0.5ml, every centrifuge tube.The LAMP reaction solution comprises 0.2 μ M F3 and B3,1.6 μ M FIP and BIP, 1M trimethyl-glycine, 4mM sal epsom, 1.0mMdNTPs, 1 * ThermoPol reaction buffer.More than the concentration of each composition be the final concentration in reaction solution.The concrete compound method of LAMP reaction solution is following:
(2) 50 μ l (400units) Bst archaeal dna polymerases (New England Biolabs, USA).
(3) developer: the optical dye SYBR GREEN I of 125 μ l 10%.
(3) LAMP detection method
The method of use of the test kit of the detection abortus Chlamydia psittaci that the present invention set up is following:
1, the extraction of trachomatis dna
With QIAamp DNA extraction test kit from abortus Chlamydia psittaci with reference to extracting total DNA strain and the pathological material of disease, the process for extracting by specification carries out.Also can adopt the commercial kit of other producers to extract DNA.
2, LAMP reaction system
3, LAMP response procedures
Earlier the LAMP reaction solution of 22 μ l and the template DNA of 2 μ l are mixed,,, add the Bst archaeal dna polymerase of 1 μ l again,, then temperature is risen to 80 ℃ and keep the 2min termination reaction at 62 ℃ of isothermal reaction 1h then at chilling on ice at 94 ℃ of reaction 5min.
4, the LAMP reaction result is judged
2.5 μ l developers are added in the reaction system,, contain abortus Chlamydia psittaci in the decidable sample if reaction solution presents green; If it is orange that reaction solution appears, then judge not contain abortus Chlamydia psittaci in the sample; Concrete like Fig. 1, be followed successively by positive control, positive, negative control and blank pipe from left to right.In addition, get 2 μ l reaction solutions and on 2% sepharose, carry out electrophoresis, in the full automatic gel imaging analysis instrument, observe electrophoresis result, the gradient band is judged to the positive if appear clearly behind the reaction product electrophoresis, otherwise is judged to feminine gender; Concrete the 1-8 swimming lane is all positive among the A like Fig. 2, and 9 is negative, and P and N then are respectively positive control and negative control.
Susceptibility to test kit among the present invention compares with the conventional sleeve type PCR method that detects miscarriage C.psittaci.The used primer of sleeve type PCR method first round amplification is ompA1 (5 ' GAG GTG AGTATG AAA AAA CTC TTG 3 ') and ompA2 (5 ' CAA GGT TGT AAT CTC TAGGTT TCA 3 '); Amplification program is: 94 ℃ of preparatory sex change 5min; Then carry out 35 circulation (94 ℃ of 30s; 52 ℃ of 1min, 72 ℃ of 2min), last 72 ℃ are extended 5min.Second take turns the amplification used primer be ompA3 (5 ' TAT GAA AAA ACT CTT GAA ATC GGC 3 ') and ompA4 (5 ' GAT AGC GGGACA AAA AGT TAG GAT 3 '); Amplification program comprises 20 circulation (94 ℃ of 30s; 50 ℃ of 1min, 72 ℃ of 1.5min).Used template is the C.psittaci SX5 strain DNA of serial dilution, 4 * 10
7, 4 * 10
6, 4 * 10
5, 4 * 10
4, 4 * 10
3, 400,40,4,0.4 copies, totally 9 gradients.Detect respectively with the template of the test kit among the present invention, also detect simultaneously, relatively the susceptibility of two kinds of methods with the sleeve type PCR method to these different copy numbers.The result shows that test kit among the present invention and sleeve type PCR method all can detect the dna profiling of 4 copies, explains that test kit and sleeve type PCR have identical susceptibility (as shown in Figure 2).
The test kit detected result shows, C.psittaci vaccine strain D13, and A and SX5 are all positive with the plasmid pMD-MOMP that contains SX5 strain MOMP gene; The plasmid Cpn0695-pGEX-6, chlamydia trachomatis, Brucella, intestinal bacteria, vibrio cholerae and the clostridium perfringens that contain CPN (C.pneumoniae) MOMP gene are all negative; The distinctive C.psittaci of the detecting abortion strain of this test kit ability is described, is had high specific.
Claims (2)
1. a test kit that detects abortus Chlamydia psittaci is characterized in that including in the test kit two pairs of Auele Specific Primers being made up of outer primer and inner primer, and wherein: the sequence of upper reaches outer primer F3 is SEQ ID № 1; The sequence of downstream outer primer B3 is SEQ ID № 2; The sequence of upper reaches inner primer FIP is SEQ ID № 3; The sequence of downstream inner primer BIP is SEQ ID № 4.
2. the method for use of the test kit of the detection abortus Chlamydia psittaci of claim 1 is characterized in that:
A. be mixed with the LAMP reaction solution with two pairs of special primers in the test kit;
B. from test sample, extract DNA;
C. be that template and LAMP reaction solution are mixed with the LAMP reaction mixture with DNA;
D. add the Bst archaeal dna polymerase at the LAMP reaction mixture and carry out the LAMP reaction;
Carry out agarose electrophoresis behind the e.LAMP reaction terminating; Can the test sample of gradient band judgement clearly be positive or negative according to appearing behind the reaction product electrophoresis; Perhaps directly visual inspection behind the adding developer in the LAMP reaction product, whether variable color judges that sample is positive or negative per sample.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087819A (en) * | 2015-09-27 | 2015-11-25 | 贵州省畜牧兽医研究所 | Ovine chlamydia LAMP detection kit |
| CN113136445A (en) * | 2021-05-20 | 2021-07-20 | 北京市朝阳区动植物疫病预防控制中心(北京市朝阳区农产品质量安全综合检测站) | LAMP primer and loop-mediated isothermal amplification detection kit for pet zoonosis chlamydia psittaci |
| CN114540525A (en) * | 2022-04-26 | 2022-05-27 | 中国人民解放军军事科学院军事医学研究院 | Primer composition for detecting or assisting in detecting chlamydia psittaci and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087819A (en) * | 2015-09-27 | 2015-11-25 | 贵州省畜牧兽医研究所 | Ovine chlamydia LAMP detection kit |
| CN113136445A (en) * | 2021-05-20 | 2021-07-20 | 北京市朝阳区动植物疫病预防控制中心(北京市朝阳区农产品质量安全综合检测站) | LAMP primer and loop-mediated isothermal amplification detection kit for pet zoonosis chlamydia psittaci |
| CN113136445B (en) * | 2021-05-20 | 2022-04-01 | 北京市朝阳区动植物疫病预防控制中心(北京市朝阳区农产品质量安全综合检测站) | LAMP primer and loop-mediated isothermal amplification detection kit for pet zoonosis chlamydia psittaci |
| CN114540525A (en) * | 2022-04-26 | 2022-05-27 | 中国人民解放军军事科学院军事医学研究院 | Primer composition for detecting or assisting in detecting chlamydia psittaci and application thereof |
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Application publication date: 20121121 |