CN102251039A - Kit and method for detecting anaplasmosis and rickettsioses based on loop-mediated isothermal amplification (LAMP) - Google Patents
Kit and method for detecting anaplasmosis and rickettsioses based on loop-mediated isothermal amplification (LAMP) Download PDFInfo
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Abstract
The invention discloses a kit and method for detecting anaplasmosis and rickettsioses based on LAMP. The invention relates to a molecular biological detection technology for anaplasmosis and rickettsioses, provides specific primers which are designed to be directed to ompB gene of spotted fever group rickettsia (SFGR) and msp2 gene of anaplasma phagocytophilum, and provides a detection kit for SFGR and anaplasma phagocytophilum by using LAMP. The kit has the characteristics of rapidness, sensitivity, high specificity, no experimental condition restrictions, etc., and is applicable to clinical detection for spotted fever and granulocytic anaplasmosis, especially in rural areas where medical conditions are limited. The invention also discloses a method for detecting rickettsioses with the kit.
Description
Technical field
The present invention relates to rickettsial detection kit and method.Particularly the present invention relates to adopt loop-mediated isothermal amplification technology (Loop-mediated isothermal amplifition LAMP) detects the test kit of pathogenic micro-organism, with and detection method.
Background technology
Loop-mediated isothermal amplification (LAMP) is a kind of new nucleic acid amplification method.The LAMP method has high sensitivity, high specific and characteristics such as easy and simple to handle, and can be under constant temperature, in one hour with goal gene amplification 10
9Doubly.Spot heat is the tick-borne rickettsiosis [P.Parola that is caused by the entozoic spot heat group's rickettsia of a class special sexual cell (SFGR), C.D.Paddock, D.Raoult, Tick-borne rickettsioses around the world:emerging diseases challenging old concepts, Clin Microbiol Rev, vol.18, no.4, pp.719-756,2005.].This disease worldwide extensively distributes.SFGR can be by propagation such as multiple media such as tick, flea and mites.Main Susceptible population is engaged in the soldier of open-air field active peasant, forest operation workman, open-air Military activity and travel staff etc.Before 1984, confirm to have only 5 kinds of spot heat group rickettsia that the people is had pathogenic effects in the world wide.Yet, have been found that ten at present surplus kind of spot heat group rickettsia.The SFGR that China exists mainly contains siberian rickettsia (R.sibirica) [L.Zhang, J.Jin, X.Fu et al., Genetic differentiation of Chinese isolates of Rickettsia sibirica by partial ompA gene sequencing and multispacer typing, Clin Microbiol, vol.44, no.7, pp.2465-2467,2006.], Heilungkiang rickettsia (R.heilongjiangensis) and dermacetor sibericus Inner Mongol subspecies (Rickettsia sibirica mongolotimonae) [D.Raoult, P.E.Fournier, M.Eremeeva et al., Naming of Rickettsiae and rickettsial diseases, Ann N Y Acad Sci, vol.1063, no.pp.1-12,2005.].The typical clinical performance for want of of such disease, easily mistaken diagnosis and delay diagnosis and treatment can cause serious disease and may cause death [A.S.Chapman, J.S.Bakken, S.M.Folk et al., Diagnosis and management of tickborne rickettsial diseases:Rocky Mountain spotted fever, ehrlichioses, and anaplasmosis--United States:a practical guide for physicians and other health-care and public health professionals, MMWR Recomm Rep, vol.55, no.RR-4, pp.1-27,2006.].The ultimate challenge of such medical diagnosis on disease at present and treatment is the laboratory early diagnosis.Serodiagnosis need acute phase and decubation paired sera, be unfavorable for early diagnosis.It is reliable laboratory diagnosis technology that pathogen separation is cultivated, but low and have only specialized laboratory to carry out because of separation rate, also is unfavorable for early diagnosis.The round pcr that with the specific gene is testing goal is the quick diagnosis technology that replaces separation and Culture at present.But quantitative fluorescent PCR needs specific apparatus, and the time-consuming and easy pollution of common Chao Shi PCR.Both detect positive rate because of mammalian DNA disturbs also can influence simultaneously.
Human granulocyte anaplasmosis (HGA) is a kind ofly to engulf the New Development tick that incorporeity (Anaplasma phagocytophilum) causes and pass the Amphixenosis by having a liking for, and nineteen ninety and 1997 are respectively at the U.S. and Europe report.This disease is 15%-36%[I.J.JW at the popular regional serum prevalence rate of America and Europe, J.I.Meek, M.L.Cartter et al., The emergence of another tickborne infection in the 12-town area around Lyme, Connecticut:human granulocytic ehrlichiosis, Infect Dis, vol.181, no.4, pp.1388-1393,2000.].Incorporeity ward infection incident [L.Zhang had taken place in Anhui Province hospital in 2006, Y.Liu, D.Ni et al., Nosocomial transmission of human granulocytic anaplasmosis in China, JAMA, vol.300, no.19, pp.2263-2270,2008.], continue after, national agriculture high risk population's incorporeity seroepidemiological survey result shows that positive rate of antibody is 13.49% (not delivering).China's rickettsiosis ultimate challenge is early stage quick diagnosis.The tradition indirect immunofluorescence method detects antibody not only needs expensive fluorescent microscope, also needs to wait patient's convalescent-stage specimen, is unfavorable for early diagnosis.The quantitative fluorescent PCR that the Chao Shi PCR of the regular-PCR that susceptibility is low, time-consuming easy pollution and susceptibility are higher etc. all needs certain instrument, and this has just limited the diagnosis of Rural areas anaplasmosis, yet, these the area and be the rickettsiosis hotspot.This just is badly in need of exploitation detection method quick, sensitive and simple to operate to be applied to the Rural areas.
This paper adopts loop-mediated isothermal amplification technology (the Loop-mediated isothermal amplifition of report in recent years, LAMP) [T.Notomi, H.Okayama, H.Masubuchi et al., Loop-mediated isothermal amplification of DNA, Nucleic Acids Res, vol.28, no.12, pp.E63,2000],, set up fast respectively with the rickettsial ompB gene of spot heat group with have a liking for that to engulf asomatous msp2 gene be the basic design primer, responsive special, economy is particularly useful for the LAMP detection kit and the detection method of above two kinds of pathogenic micro-organisms of vast Rural areas.
Summary of the invention
The objective of the invention is at existing diagnostic method consuming time, need specific apparatus, and sensitivity and specificity are not high, providing a kind of is used to detect spot heat group rickettsia and has a liking for and engulf asomatous high specificity, the susceptibility height, rapid reaction, easy and simple to handle, and do not need the detection method and the test kit of specific apparatus, can better be applied to the insufficient regional clinical diagnosis of condition equipment.
The invention discloses the rickettsial test kit of a kind of employing LAMP technology for detection spot heat group, comprise the LAMP reaction solution that detects the ompB gene, described LAMP reaction solution contains: 6 Auele Specific Primers, damping fluid, dNTP mixed solution, MgSO
4Solution, betaine solution, archaeal dna polymerase.
The present invention discloses a kind of employing LAMP technology for detection and have a liking for and engulf asomatous test kit, comprise the LAMP reaction solution that detects the msp2 gene, described LAMP reaction solution contains: 6 Auele Specific Primers, damping fluid, dNTP mixed solution, MgSO
4Solution, betaine solution, archaeal dna polymerase.
The invention also discloses the application of described test kit in preparation detection rickettsia instrument, its use comprises following process:
1) at first, will comprise Auele Specific Primer, damping fluid, dNTP mixed solution, betaine solution, MgSO
4The LAMP reaction solution of solution and archaeal dna polymerase mixes with detected sample.
2) secondly, reaction system is placed 61-65 ℃ of isothermal reaction 0.5-2h, 80-90 ℃ of constant temperature termination reaction 3-15min.
3) carry out reaction product at last and detect, reaction product is carried out agarose gel electrophoresis detect; Or the SYBR Green I that in reaction tubes, adds, by observing the color judged result, leave standstill 2-10min after, greeny positive in the reaction tubes, be orange negative.
The rickettsial test kit of employing LAMP technology for detection spot heat group of the present invention comprises the LAMP reaction solution, comprises 6 Auele Specific Primers in the described reaction solution.
The design of the rickettsial LAMP Auele Specific Primer of spot heat group: by sequence analysis software 17 ompB gene orders among the Genbank are compared, according to 8 zone design 6 primers F IP-1, BIP-1, F3-1, B3-1, LFA-1, the LBA-1 of Rickettsia sp.BJ-90 (Genbank:AY331393).A part or its complementary strand complementation of 1209～1427 nucleotide sequence of described primer and target gene.The sequence of described primers F 3-1 is Sequence NO.1, and the sequence of primer B3-1 is that the sequence of Sequence NO.2, primers F IP-1 is that the sequence of Sequence NO.3, primer BIP-1 is that the sequence of Sequence NO.4, primer LFA-1 is that the sequence of Sequence NO.5, primer LBA-1 is Sequence NO.6.I in the described sequence is that xanthoglobulin can match with cytosine(Cyt) and VITAMIN B4.
Perhaps include holding or/and the sequence that 3 ' end prolongs of above-mentioned sequence to 5 ';
Perhaps with above-mentioned sequence homology greater than 85% sequence;
The perhaps complementary sequence of above-mentioned sequence.
Detection spot heat group rickettsia ompB LAMP detection kit provided by the invention also includes the LAMP reaction solution of 6 primers, constitutes the LAMP detection architecture jointly.
The specific configuration of 25 μ L ompB LAMP detection architecture is as follows: FIP and each 1.6uM of BIP primer, LF and each 0.8uM of LB primer, F3 and each 0.2uM of B3 primer, 20mM Tris-HCl (pH 8.8), 10mM KCl, 8mM MgSO
4, 10mM (NH
4)
2SO
4, 0.1%Tween 20,0.8M betaine and dNTPs 1.4mM, 1 μ l Bst DNA enzyme (8U/ μ l).
The detection reaction of detection spot heat group rickettsia ompB LAMP test kit of the present invention, 63 ℃ of isothermal reaction 1h of elder generation, 80 ℃ of termination reaction 5min.Detect in 2% agarose gel electrophoresis by 3 μ L reaction product, or in reaction tubes, add 1000 * SYBR Green I of 1 μ L, by observing the color judged result, leave standstill 3～5min after, greeny positive in the reaction tubes, be orange negative.
The present invention adopts the LAMP technology for detection to have a liking for to engulf asomatous test kit, comprise and have a liking for the design of engulfing asomatous LAMP Auele Specific Primer: by sequence analysis software 82 msp2 gene orders among the Genbank are compared, according to 8 zone design 6 primers F IP-2, BIP-2, F3-2, B3-2, LFA-2, the LBA-2 of A.phagocytophilum isolate EQ6 D22 (Genbank:AY763471).A part or its complementary strand complementation of 374～592 nucleotide sequence of described primer and target gene.The sequence of described primers F IP-2 is Sequence NO.7, and the sequence of primer BIP-2 is that the sequence of Sequence NO.8, primers F 3-2 is that the sequence of Sequence NO.9, primer B3-2 is that the sequence of Sequence NO.10, primer LFA-2 is that the sequence of Sequence NO.11, primer LBA-2 is Sequence NO.12.I in the described sequence is that xanthoglobulin can match with cytosine(Cyt) and VITAMIN B4.
Perhaps include holding or/and the sequence that 3 ' end prolongs of above-mentioned sequence to 5 ';
Perhaps with above-mentioned sequence homology greater than 85% sequence;
The perhaps complementary sequence of above-mentioned sequence.
Msp2LAMP detection kit provided by the invention also includes the LAMP reaction solution of 6 primers, constitutes the LAMP detection architecture jointly.
The specific configuration of 25 μ L msp2LAMP detection architecture is as follows: the specific configuration of 25 μ L ompB LAMP detection architecture is as follows: FIP and each 1.6uM of BIP primer, LF and each 0.8uM of LB primer, F3 and each 0.2uM of B3 primer, 20mM Tris-HCl (pH 8.8), 10mM KCl, 8mM MgSO
4, 10mM (NH
4)
2SO
4, 0.1%Tween 20,0.8M betaine and dNTPs 1.4mM, 1 μ l BstDNA enzyme (8U/ μ l).
The detection reaction of msp2LAMP test kit of the present invention, 63 ℃ of isothermal reaction 1h of elder generation, 80 ℃ of termination reaction 5min.Detect in 2% agarose gel electrophoresis by 3 μ L reaction product, or in reaction tubes, add 1000 * SYBR Green I of 1 μ L, by observing the color judged result, leave standstill 3～5min after, greeny positive in the reaction tubes, be orange negative.
Compared with prior art, test kit provided by the invention and detection method can detect spot heat group rickettsia fast, delicately and have a liking for and engulf incorporeity, have higher specificity and testing conditions are required low, are particularly useful for the Rural areas.
Description of drawings
The rickettsial specificity LAMP primer screening of Fig. 1 spot heat group.With the three cover primers ompB genes that increase respectively, use LMAP turbidimeter LA-320 monitoring reaction, wherein the primer amplification time of B group short, the amplification amount is big.
Fig. 2 two cover ompB gene ring primers are to goal gene expanding effect figure.4 upstream and downstream primers of 2 collar primers cross match is to the expanding effect of ompB.4 groups of primer amplification efficient are more or less the same, but best with Sequence NO.5, Sequence NO.6 primer amplification effect.
The agarose electrophoresis figure of the susceptibility test that Fig. 3 spot heat group rickettsia LAMP detects.LMAP: the gene that dilutes plasmid with the primer amplification of ompB LAMP method.PCR: with the gene of standard plasmid amplification dilution plasmid.The primer of LAMP method is sensitive more.
The real-time quantitative figure of the susceptibility test that Fig. 4 spot heat group rickettsia LAMP detects.The primer of LAMP method just can detect the existence of ompB gene when 5 clones are arranged in system.
Fig. 5 has a liking for and engulfs asomatous LAMP primer screening.With the three cover primers msp2 genes that increase respectively, use the reaction of LMAP turbidimeter LA-320 instrument monitoring, wherein the primer amplification time of B group short, the amplification amount is big.
Fig. 6 two cover msp2 gene ring primers are to goal gene expanding effect figure.4 upstream and downstream primers of 2 collar primers cross match is to the expanding effect of msp2.4 groups of primer amplification efficient are more or less the same, but best with Sequence NO.11, Sequence NO.12 primer amplification effect.
Fig. 7 has a liking for the real-time quantitative figure of the susceptibility test of engulfing incorporeity LAMP detection.The primer of LAMP method just can detect the existence of msp2 gene when having 25 clones in system.
Fig. 8 has a liking for the agarose electrophoresis figure of the susceptibility test of engulfing incorporeity LAMP detection.LMAP: the gene that dilutes plasmid with the primer amplification of msp2LAMP method.PCR: with the gene of standard plasmid amplification dilution plasmid.The primer of LAMP method is sensitive more.
Embodiment
Embodiment 1: the rickettsial detection of spot heat group
1. the LAMP design of primers of spot heat group rickettsia ompB gene
Retrieval obtains rickettsia ompB gene order from Genbank, use DNAstar software to compare, conservative region at the comparison gained, with Rickettsia sp.BJ-90 (AY331393) sequence is template, use LAMP primer-design software (Primer Explorer software, version 4.0) carry out the LAMP design of primers in this zone, design 3 cover primers:
The LAMP Auele Specific Primer of table 1 spot heat group rickettsia ompB gene
It is synthetic that designed primer is served Hai Shenggong, uses the HPLC purifying.
2. the rickettsial standard plasmid preparation of spot heat group
According to 3 cover LAMP primer regions, with Rickettsia sp.BJ-90 (GenBank:AY331393) sequence is template, use Primer Premier 5 software designs 2 cover primers, production standard plasmid: OPBC1 (5 '-ATAATGGCGCAGGTAGAGC-3 ') and OPBC2 (5 '-CACCGCCAGCGTTCCCTAT-3 '); OPA1 (5 '-AACTGCTGAAATGGCTGGAC-3 ') and OPA2 (5 '-AGTCCTGCCATTGGAGTTA-3 ').It is synthetic that designed primer is served Hai Shenggong, desalting and purifying.The PCR product is 624bp, comprises 3 cover LAMP primer regions.The PCR product is used pEASY-T1Cloning Kit, and (Transgen China) connects into the pEASY-T1 carrier.After connection transforms successfully, extract plasmid, and (IMPLEN, German) instrument carries out quantitatively, afterwards with plasmid gradient dilution (5 to use NanoPhotometer
5, 5
4, 5
3, 5
2, 5
1With 5
0Copies/ μ l) to assess the minimum detectability and the repeatability of LAMP method.
3. the rickettsial specificity LAMP primer screening of spot heat group
With 5
5Copies/ μ L is a template, and distilled water is done negative control, test 3 cover LAMP primer amplification efficient.The specific configuration of 25 μ L ompB LAMP detection architecture is as follows: the specific configuration of 25 μ L ompB LAMP detection architecture is as follows: FIP/FIP-1 and each 1.6uM of BIP/FIP-1 primer, each 0.8 uM of LB and LF primer, F3/F3-1 and each 0.2uM of B3/B3-1 primer, 20mM Tris-HCl (pH 8.8), 10mM KCl, 8mM MgSO
4, 10mM (NH
4)
2SO
4, 0.1%Tween 20,0.8M betaine and dNTPs 1.4mM, 1 μ l Bst DNA enzyme (8U/ μ l).1 μ l 5
5Copies/ μ l plasmid solution.63 ℃ of isothermal reaction 1h, 80 ℃ of termination reaction 5min.Use the reaction of LMAP Turbidimeter LA-320 instrument monitoring.
From instrument monitoring result (accompanying drawing 1), can be observed, no matter from appearance time or amplification amount, the amplification efficiency maximum of B cover primer, so, select B cover primer.
Behind definite B cover primer,, use LAMP primer-design software (Primer Explorer software, version 4.0) design 2 collar primers based on B cover primer:
The rickettsial specificity LAMP primer screening of table 2 spot heat group
Equally with 5
5Copies/ μ l is a template, and distilled water is done negative control, test 3 cover LAMP primer amplification efficient.The specific configuration of 25 μ L LAMP detection architecture is as follows: the specific configuration of 25 μ L ompB LAMP detection architecture is as follows: FIP/FIP-1 and each 1.6uM of BIP/BIP-1 primer, LF/LFA-1 and each 0.8uM of LB/LBA-1 primer, F3/F3-1 and each 0.2uM of B3/B3-1 primer, 20mM Tris-HCl (pH 8.8), 10mM KCl, 8mM MgSO
4, 10mM (NH
4)
2SO
4, 0.1%Tween 20,0.8M betaine and dNTPs 1.4mM, 1 μ l Bst DNA enzyme (8U/ μ l).1 μ l 5
5Copies/ μ l plasmid solution.63 ℃ of isothermal reaction 1h, 80 ℃ of termination reaction 5min.Use the reaction of LMAP turbidimeter LA-320 instrument monitoring.
In two collar primers, intersect and detect, and be divided into 4 groups: LFA-1+LBA-1, LFA-1+LBB, LFB+LBA-1, LFB+LBB.
Can be observed from instrument monitoring result (accompanying drawing 2), the reaction efficiency of 4 groups of combination of primers is more or less the same, but organizing the composite reaction efficient of LFA-1 and LBA-1, makes up A a little more than other, so, select A group ring primer LFA-1 and LBA-1.
Through the optimization of above-mentioned experiment, thus select primers F IP-1, BIP-1, F3-1, B3-1, LFA-1, LBA-1 totally 6 primers as the Auele Specific Primer that detects spot heat group rickettsia test kit.
4. the susceptibility test of spot heat group rickettsia LAMP detection
Use the plasmid (5 of gradient dilution
5, 5
4, 5
3, 5
2, 5
1With 5
0Copies/ μ l) susceptibility of detection ompB LAMP method.Use standard plasmid primer OPBC1 and OPBC2 to carry out the PCR detection sensitivity simultaneously.The LAMP minimum detectability is 5copies/ μ l, and the PCR minimum detectability is 625copies/ μ l, and the detection sensitivity of LAMP method is at least than PCR method high 125 times (accompanying drawing 3 and 4).
5. the reperformance test of spot heat group rickettsia LAMP detection
The standard plasmid of gradient dilution detects by two kinds of methods: 1. carrying out 5 tests on the same day, and adding up each concentration plasmid and reflect peak time; 2. in 5 days, carry out a LAMP reaction respectively, add up each concentration plasmid and reflect peak time.Data will be calculated the variation within batch coefficient and the official written reply variation coefficient by SAS software (9.1 editions).
The reperformance test that table 3 spot heat group rickettsia LAMP detects
| Concentration (copies/ μ l) | CVi％ | CVo％ |
| 5 5 | 1.20％ | 2.22％ |
| 5 4 | 1.40％ | 2.53％ |
| 5 3 | 1.55％ | 2.81％ |
| 5 2 | 1.58％ | 3.11％ |
6. the specificity test of spot heat group rickettsia LAMP detection
In this research, use 42 bacterial strains to carry out the LAMP specific detection altogether, comprising the Rickettsiales member: Rickettsia prowazekii, Rickettsia typhi, Orientia tsutsugamushi type Karp, Kato and Gilliam, Rickettsia sibirica, Rickettsia conorii, Rickettsia marmionii, Rickettsia akari, Rickettsia rickettsii, Rickettsia africa, Rickettsia parkeri, Rickettsia japanica, Rickettsia slovaca, Rickettsia aeschlimannii, Rickettsia montanensis, Rickettsia helvetica, Rickettsia felis, Rickettsia australis, Rickettsia canadensis, Rickettsia bellii, Rickettsia heilongjiangeneis, Anaplasma phagocytophilium, Ehrlichia chaffeenisis and relevant bacterial strain Coxiella burnetii, Bartonella henselae, Bartonella quintana.By rickettsia and WHO cooperation center provide (Marseille, France).All bacterial strains use QIAamp DNA Mini Kit, and (Qiagen, Hilden Germany) extract genomic dna from cell culture.The genomic dna of other common clinical pathogenic bacteria, comprise: Borrelia burgdorferi, Escherichia coli, Vibrio cholerae, Bacillus anthracis, Haemophilus influenzae, Bacterium pestis, Listeria spp., Legionella spp., Yersinia pestis, Shigella dysenteriae, Neisseria meningitides, Leptospira spp., Borrelia burgdorferi, Mycobacterium tuberculosis and Klebsiella pneumoniae, infecting the section office that are correlated with by Chinese Disease Control and Prevention Center provides.In addition, from the blood of people, ox, horse, goat and the mouse of health, extract genomic dna as negative control.The genomic dna that above bacterial strain extracts at first carries out PCR by the general 16S primer of prokaryotic organism and detects validity.
In the specific detection, all samples, seven bacterial classification R.sibirica, R.conorii, R.rickettsii, R.africa, R.parkeri, R.japonica, R.heilongjiangensis detects positive, and all the other all detect feminine gender, and the specificity that proves this LAMP method is 100%.
In this research, we have obtained 11 routine rickettsia suspected case samples from 4 tame hospitals (first hospital of Beijing University, First People's Hospital, Laizhou, Beijing Friendship Hospital and the 302 hospital of 3 Chinese People's Liberation Army) in the period of 2007-2009.The suspected case sample is by rickettsia serum diagnosis standard (4 times of risings of IgG antibody titers), and the amplification of rickettsia 16SrRNA nest-type PRC detects, to get rid of the possibility of other clinical febrile diseases.Suspected case patient does not have antithrombotics blood at morbidity acute phase extraction 2ml EDTA anticoagulation and 2ml, anticoagulation is used for cause of disease and cultivates, non-anticoagulation separation of serum is used for IgM and IgG antibody test, and residue blood extracts DNA soon and is used for nest-type PRC, and the LAMP method detects in this research.Extract once more decubation patient and to carry out the IgG antibody test.The LAMP clinical assessment uses the 25ul system, uses 8ul to extract genomic dna as template.
The hot rickettsia LAMP of table 4 spot detects
Annotate: Pos: expression is positive; Neg: feminine gender
In the clinical suspected case of collecting, there are 7 examples to be diagnosed as spot heat group rickettsial infection, wherein 2 examples are positive for the rickettsia cultivation, and 5 examples are the serodiagnosis case, and both IgG antibody 4 times of risings occur than acute phase serum antibody in convalescent phase serum.In order to assess the practicality of LAMP method, we have compared nest-type PRC simultaneously.In clinical sample detected, the LAMP method detected 8 routine sample result altogether, wherein detected the 5 example positives in 7 routine confirmed cases, detected the 3 example positives in 4 routine suspected cases.And nest-type PRC does not detect the positive in clinical sample.
Embodiment 2 has a liking for and engulfs incorporeity and detect
1. have a liking for and engulf incorporeity msp2 gene LAMP design of primers
Retrieval obtains incorporeity msp2 gene order from Genbank, use DNAstar software to compare, conservative region at the comparison gained, with A.phagocytophilum isolate EQ6 D22 (AY763471) sequence is template, use LAMP primer-design software (Primer Explorer software, version 4.0) carry out the LAMP design of primers in this zone, design 3 cover primers:
Table 5 is had a liking for and is engulfed incorporeity msp2 gene LAMP design of primers
It is synthetic that designed primer is served Hai Shenggong, uses the HPLC purifying.
2. have a liking for and engulf the preparation of incorporeity msp2 detection gene standard plasmid
According to 3 cover LAMP primer regions, with A.phagocytophilum isolate EQ6 D22 (AY763471) sequence is template, use Primer Premier 5 software designs 1 cover primer, production standard matter: MP1 (5 '-GAAGCGTAATGATGTCTA-3 ') and reverse primer MP2 (5 '-AGTAACAACATCATAAGC-3 ').It is synthetic that designed primer is served Hai Shenggong, desalting and purifying.The PCR product is 452bp, comprises 3 cover LAMP primer regions.The PCR product is used pEASY-T1 Cloning Kit, and (Transgen China) connects into the pEASY-T1 carrier.After connection transforms successfully, extract plasmid, and use NanoPhotometer (IMPLEN, German) instrument carries out quantitatively, afterwards, with plasmid gradient dilution (5
5, 5
4, 5
3, 5
2, 5
1With 5
0Copies/ μ l) to assess the minimum detectability and the repeatability of LAMP method.
3. have a liking for and engulf asomatous LAMP primer screening
With 5
5Copies/ μ l is a template, and distilled water is done negative control, test 3 cover LAMP primer amplification efficient.The specific configuration of 25 μ L msp2 LAMP detection architecture is as follows: FIP/FIP-2 and each 1.6uM of BIP/BIP-2 primer, LF and each 0.8uM of LB primer, each primer 0.2uM of F3/F3-2 and B3/B3-2,20mMTris-HCl (pH 8.8), 10mM KCl, 8mM MgSO
4, 10mM (NH
4)
2SO
4, 0.1%Tween 20,0.8M betaine, and 1.4mM dNTPs, 1 μ l Bst DNA enzyme (8U/ μ l), 1 μ l 5
5Copies/ μ l plasmid solution.63 ℃ of isothermal reaction 1h, 80 ℃ of termination reaction 5min.Use the reaction of LMAP turbidimeter LA-320 instrument monitoring.
Can be observed from instrument monitoring result (accompanying drawing 5), no matter from appearance time or amplification amount, the amplification efficiency of B cover primer is the highest, so, select B cover primer.
Behind definite B cover primer,, use LAMP primer-design software (Primer Explorer software, version 4.0) design 2 collar primers based on B cover primer:
Table 6 is had a liking for and is engulfed asomatous LAMP primer screening
Equally with 5
5Copies/ μ l is a template, and distilled water is done negative control, test 3 cover LAMP primer amplification efficient.25 μ L msp2LAMP detection architecture specific configurations are as follows: FIP/FIP-2 and each 1.6uM of BIP/BIP-2 primer, LF and each 0.8uM of LB primer, F3/F3-2 and each 0.2uM of B3/B3-2 primer, 20mM Tris-HCl (pH 8.8), 10mM KCl, 8mM MgSO
4, 10mM (NH4)
2SO
4, 0.1%Tween 20,0.8M betaine, and 1.4mM dNTPs, 1 μ l Bst DNA enzyme (8U/ μ l), 1 μ l 5
5Copies/ μ l plasmid solution.63 ℃ of isothermal reaction 1h, 80 ℃ of termination reaction 5min.Use the reaction of LMAP turbidimeter LA-320 instrument monitoring.In two collar primers, intersect and detect, and be divided into 4 groups: LFA-2+LBA-2, LFA-2+LBB, LFB+LBA-2, LFB+LBB.
Can be observed from instrument monitoring result (accompanying drawing 6), the reaction efficiency of 4 groups of combination of primers is more or less the same, but organizing the composite reaction efficient of LF and LB, makes up A a little more than other, so, select A group ring primer LFA-2 and LBA-2.
Through the optimization of above-mentioned experiment, so select primers F IP-2, BIP-2, F3-2, B3-2, LFA-2, LBA-2 totally 6 Auele Specific Primers that primer is had a liking for to engulf the incorporeity test kit as detection.
4. have a liking for and engulf the test of incorporeity LAMP detection sensitivity
Use the plasmid (5 of gradient dilution
5, 5
4, 5
3, 5
2, 5
1And 5
0Copies/ μ l) susceptibility of detection msp2LAMP method.Use standard plasmid primer MP1 and MP2 to carry out the PCR detection sensitivity simultaneously.The LAMP minimum detectability is 25copies/ μ l, and the PCR minimum detectability is 625copies/ μ l, and the detection sensitivity of LAMP method is at least than PCR method high 25 times (accompanying drawing 7 and 8).
5. have a liking for and engulf incorporeity LAMP detection reperformance test
The gradient dilution standard plasmid detects by two kinds of methods: 1. carrying out 5 tests on the same day, and adding up each concentration plasmid and reflect peak time; 2. in 5 days, carry out a LAMP reaction respectively, add up each concentration plasmid and reflect peak time.Data will be calculated the variation within batch coefficient and the official written reply variation coefficient by SAS software (9.1 editions).
Table 7 is had a liking for the reperformance test of engulfing incorporeity LAMP detection
6. have a liking for and engulf the test of incorporeity LAMP detection specificity
In this research, use 42 bacterial strains to carry out the LAMP specific detection altogether, comprising the Rickettsiales member: Rickettsia prowazekii, Rickettsia typhi, Orientia tsutsugamushi type Karp, Kato and Gilliam, Rickettsia sibirica, Rickettsia conorii, Rickettsia marmionii, Rickettsia akari, Rickettsia rickettsii, Rickettsia africa, Rickettsia parkeri, Rickettsia japanica, Rickettsia slovaca, Rickettsia aeschlimannii, Rickettsia montanensis, Rickettsia helvetica, Rickettsia felis, Rickettsia australis, Rickettsia canadensis, Rickettsia bellii, Rickettsia heilongjiangeneis, Anaplasma phagocytophilium, Ehrlichia chaffeenisis and relevant bacterial strain Coxiella burnetii, Bartonella henselae, Bartonella quintana.By rickettsia and WHO cooperation center provide (Marseille, France).All bacterial strains use QIAamp DNA Mini Kit, and (Qiagen, Hilden Germany) extract genomic dna from cell culture.The genomic dna of other common clinical pathogenic bacteria, comprise: Borrelia burgdorferi, Escherichia coli, Vibrio cholerae, Bacillus anthracis, Haemophilus influenzae, Bacterium pestis, Listeria spp., Legionella spp., Yersinia pestis, Shigella dysenteriae, Neisseria meningitides, Leptospira spp., Borrelia burgdorferi, Mycobacterium tuberculosis and Klebsiella pneumoniae, infecting the section office that are correlated with by Chinese Disease Control and Prevention Center provides.In addition, from the blood of people, ox, horse, goat and the mouse of health, extract genomic dna as negative control.The genomic dna that above bacterial strain extracts at first carries out PCR by the general 16S primer of prokaryotic organism and detects validity.
In the specific detection, all samples, except that A.phagocytophilium detected the positive, all the other all detected feminine gender, and the specificity that proves this LAMP method is 100%.
7. have a liking for and engulf the clinical assessment that incorporeity LAMP detects
In this research, we have obtained 42 routine incorporeity suspected case samples from 4 tame hospitals (first hospital of Beijing University, First People's Hospital, Laizhou, Beijing Friendship Hospital and the 302 hospital of 3 Chinese People's Liberation Army) in the period of 2007-2009.The suspected case sample is by incorporeity serum diagnosis standard (4 times of risings of IgG antibody titers), and amplification of incorporeity 16SrRNA nest-type PRC or incorporeity msp2 gene by fluorescence quantitative pcr amplification detect, to get rid of the possibility of other clinical febrile diseases.Suspected case patient does not have antithrombotics blood at morbidity acute phase extraction 2mlEDTA anticoagulation and 2ml, anticoagulation is used for cause of disease and cultivates, no antithrombotics blood system is used for IgM and IgG antibody test from serum, and the residue clot extracts DNA and is used for nest-type PRC, and the LAMP method detects in quantitative fluorescent PCR and this research.Extract no antithrombotics blood separation serum decubation once more patient and carry out the IgG antibody test.The LAMP clinical assessment uses the 25ul system, uses 8ul to extract genomic dna as template.
Table 8 is had a liking for the clinical assessment of engulfing incorporeity LAMP detection
Annotate: Pos: expression is positive; Neg: feminine gender
In the clinical suspected case of collecting, in this breadboard detection, have 15 examples to be diagnosed as incorporeity and infect, wherein 2 examples are cultivated positively for incorporeity, 13 examples are serodiagnosis, at convalescent phase serum IgG antibody than 4 times of risings of acute phase antibody appearance.In order to assess the practicality of LAMP method, we have compared nest-type PRC and real-time PCR simultaneously.In the detection of clinical sample, the LAMP method detects the 26 routine samples result that is positive altogether, and it is positive wherein to detect 12 examples in 15 routine confirmed cases, and it is positive to detect 14 examples in 27 routine suspected cases.The 3 example positives (P＜0.001) that this result is detected apparently higher than detected 1 example positive (P＜0.001) of nest-type PRC and real-time PCR.
Claims (5)
1. one kind is adopted the rickettsial test kit of LAMP technology for detection spot heat group, comprises the LAMP reaction solution that detects the ompB gene, and described LAMP reaction solution contains: 6 Auele Specific Primers, damping fluid, dNTP mixed solution, MgSO
4Solution, Betaine solution, archaeal dna polymerase.
2. LAMP reaction solution according to claim 1, it is characterized in that the sequence of described 6 Auele Specific Primers is: Sequence NO.1, Sequence NO.2, Sequence NO.3, Sequence NO.4, Sequence NO.5, Sequence NO.6.
3. one kind is adopted the LAMP technology for detection to have a liking for to engulf asomatous test kit, comprise the LAMP reaction solution that detects the msp2 gene, and described LAMP reaction solution contains: 6 Auele Specific Primers, damping fluid, dNTP mixed solution, MgSO
4Solution, Betaine solution, archaeal dna polymerase.
4. LAMP reaction solution according to claim 3, it is characterized in that the sequence of described 6 Auele Specific Primers is: Sequence NO.7, Sequence NO.8, Sequence NO.9, Sequence NO.10, Sequence NO.11, Sequence NO.12.
5. the described test kit of claim 1-4 detects spot heat group's rickettsia and has a liking for the application of engulfing in the incorporeity instrument in preparation, and its use comprises following process:
1) at first, will comprise Auele Specific Primer, damping fluid, dNTP mixed solution, Betaine solution, MgSO
4The LAMP reaction solution of solution and archaeal dna polymerase mixes with detected sample.
2) secondly, reaction system is placed 61-65 ℃ of isothermal reaction 0.5-2h, 80-90 ℃ of constant temperature termination reaction 3-15min.
3) carry out reaction product at last and detect, reaction product is carried out agarose aggegation electrophoresis detection; Or the SYBR Green I that in reaction tubes, adds, by observing the color judged result, leave standstill 2-10min after, greeny positive in the reaction tubes, be orange negative.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102936622A (en) * | 2012-09-28 | 2013-02-20 | 中国检验检疫科学研究院 | Non-diagnostic method for detecting rickettsia sibirica through loop-mediated isothermal amplification technology |
| CN110468222A (en) * | 2019-09-16 | 2019-11-19 | 中国人民解放军军事科学院军事医学研究院 | The special primer for checking of one group of China's Rana amurensis |
| KR102258934B1 (en) * | 2021-01-26 | 2021-06-01 | 주식회사 에이아이더뉴트리진 | Composition for diagnosis of Lyme disease and kit comprising the same |
| CN114250311A (en) * | 2022-02-09 | 2022-03-29 | 国科宁波生命与健康产业研究院 | CDA primer group and kit for detecting spotted fever group rickettsia and application of CDA primer group and kit |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695538B (en) * | 2013-12-05 | 2016-08-17 | 河南农业大学 | Use LAMP technology detection Anaplasma phagocytophilum method |
-
2011
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Non-Patent Citations (2)
| Title |
|---|
| QUAN LIN等: "《Anaplasma phagocytophilum Has a Functional msp2 Gene That Is Distinct from p44》", 《INFECTION AND IMMUNITY》 * |
| ROBERT F.MASSUNG等: "《Comparison of PCR Assays for Detection of the Agent of Human Granulocytic Ehrlichiosis, Anaplasma phagocytophilum》", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102936622A (en) * | 2012-09-28 | 2013-02-20 | 中国检验检疫科学研究院 | Non-diagnostic method for detecting rickettsia sibirica through loop-mediated isothermal amplification technology |
| CN110468222A (en) * | 2019-09-16 | 2019-11-19 | 中国人民解放军军事科学院军事医学研究院 | The special primer for checking of one group of China's Rana amurensis |
| CN110468222B (en) * | 2019-09-16 | 2022-08-30 | 中国人民解放军军事科学院军事医学研究院 | Special detection primers for rickettsiae in China Heilongjiang |
| KR102258934B1 (en) * | 2021-01-26 | 2021-06-01 | 주식회사 에이아이더뉴트리진 | Composition for diagnosis of Lyme disease and kit comprising the same |
| WO2022163967A1 (en) * | 2021-01-26 | 2022-08-04 | 주식회사 에이아이더뉴트리진 | Composition for diagnosing lyme disease and kit comprising same |
| CN114250311A (en) * | 2022-02-09 | 2022-03-29 | 国科宁波生命与健康产业研究院 | CDA primer group and kit for detecting spotted fever group rickettsia and application of CDA primer group and kit |
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| CN102251039B (en) | 2013-04-03 |
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