CN105238860A - LAMP (loop-mediated isothermal amplification) kit for detection of mycoplasma pneumoniae and special LAMP primer for detection of mycoplasma pneumoniae - Google Patents
LAMP (loop-mediated isothermal amplification) kit for detection of mycoplasma pneumoniae and special LAMP primer for detection of mycoplasma pneumoniae Download PDFInfo
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Abstract
The invention discloses a LAMP (loop-mediated isothermal amplification) kit for detection of mycoplasma pneumoniae and a special LAMP primer for detection of mycoplasma pneumoniae. The special LAMP primer for detection of mycoplasma pneumoniae is designed according to a specificity conservative target sequence of a mycoplasma pneumoniae P1 gene (GenBank number: CP002077.1). The LAMP primer is formed by six primers including outer primers MP-16F3 and MP-16B3, inner primers MP-16FIP and MP-16BIP and loop primers MP-16LF and MP-16LB. By the aid of the LAMP kit and the special LAMP primer for detection of mycoplasma pneumoniae, quickness, convenience, high efficiency, high specificity and high sensitivity in qualitative detection of the mycoplasma pneumoniae in samples of pure bacteria, sputum, bronchoalveolar lavage fluid, throat swabs and the like can be realized without complicated instruments, and a new technical platform is provided for detection of the mycoplasma pneumoniae.
Description
Technical field
The invention belongs to the molecular Biological Detection of bacterium in biological technical field, particularly relate to a kind of LAMP kit of mycoplasma pneumoniae and primer special and its thereof and detecting the application in mycoplasma pneumoniae.
Background technology
Mycoplasma pneumoniae (Mycoplasmapneumoniae, Mp) is one of modal pathogenic bacterium of respiratory tract infection.China epidemiology survey display Mp has become the primary pathogenic bacterium of China's Adults Community acquired pneumonia.Mp infects not only can cause respiratory tract disease, and also can cause whole body multiple organ pathology, as encephalitis, myocarditis, hepatitis, ephritis, immune hemolytic anemia etc., and the critical case that Mp caused in the last few years is on the increase.Because Mp lacks cell walls, as invalid in beta-lactam etc. to common antibiotics.Therefore, timely clear and definite Mp infects significant to the timely control of antibiotic choose reasonable and infection.
Ring mediated constant temperature nucleic acid amplification technology (1oop-mediatedisothermalamplification, LAMP) be by Notomi (NotomiT, etal.Loop-mediatedisothermalamplificationofDNA.NucleicAc idsRes2000; 28 (12): 63.) a kind of novel gene amplification technique invented.Concrete grammar is 6 regions for target gene (DNA or cDNA), design 4-6 species-specific primers, utilize strand displacement archaeal dna polymerase, under isothermal conditions can efficiently, fast, high amplified target sequence specifically, result directly judges by the precipitation turbidity of the by product magnesium pyrophosphate that increases.LAMP technology has rapidly and efficiently, high specificity, highly sensitive, simple to operate, detect the features such as directly perceived and equipment requirements is low.
Since invention in 2000, LAMP technology is by the rapid molecular Biological Detection field for fields such as the pathogenic microorganism examination, genetic diseases diagnosis, food safeties.In recent years, this technology is widely used in pathogen detection abroad.The people such as MasakiImai (ImaiM, NinomiyaA, MinekawaHRapiddiagnosisofH5N1avianinfluenzavirusinfectio nbynewlydevelopedinfluenzaH5hemagglutiningene-specificlo op-mediatedisothermalamplificationmethod. [J] VirolMethods.2007May; 141 (2): 173-80.) LAMP is utilized to establish the Laboratory Diagnosed system of avian influenza virus.The people such as NobuyukiHayashi (HayashiN, AraiR, TadaS, TaguchiH, OgawaYFoodMicrobiol.2007Oct-Dec; ITS sequence for four kind cordiale yeast devise LAMP Auele Specific Primer, establishes efficient LAMP detection system 24 (7-8): 778-85DetectionandidentificationofBrettanomyces/Dekkeras p.yeastswithaloop-mediatedisothermalamplificationmethod.).The LAMP detection method set up based on LAMP technology can detect other virus relevant to the mankind, as Viral Hemorrhagic septicemia (VHS), cytomegalovirus (CMV), Ebola virus (EBOV), chronic burkitt's lymphoma virus (EBV), irido virus, mankind's herpus vivus 8 type, haematopoietic necrosis virus (HHNV), tomato spotted wilf virus, tomato yellow leaf curl virus etc.At present, domestic there are no the LAMP kit for detecting mycoplasma pneumoniae and primer special thereof.
Summary of the invention
The invention provides the primer for carrying out LAMP detection to mycoplasma pneumoniae, to realize the batch detection of mycoplasma pneumoniae, improve the specificity and sensitivity that detect.
LAMP primer for detecting mycoplasma pneumoniae provided by the present invention, design according to specific conservative's target sequence of mycoplasma pneumoniae P1 gene (No. GenBank: CP002077.1), in order to the mycoplasma pneumoniae in the samples such as the pure bacterium of qualitative detection, sputum, bronchoalveolar lavage fluid, throat swab, described LAMP primer is made up of six primers, comprises the combination of outer primer MP-16F3 and MP-16B3, inner primer MP-16FIP and MP-16BIP and ring primer MP-16LF and MP-16LB; Specific conservative's target sequence of described mycoplasma pneumoniae pneumonia mycoplasma P1 gene is as shown in SEQ ID NO:1.
Specifically, the nucleotide sequence of described six primers for carrying out LAMP detection to mycoplasma pneumoniae is as shown in SEQ ID NO:2 (MP-16F3), SEQIDNO:3 (MP-16B3), SEQIDNO:4 (MP-16FIP), SEQIDNO:5 (MP-16BIP), SEQIDNO:6 (MP-16LF) and SEQIDNO:7 (MP-16LB).
Described LAMP primer is MP-16FIP and MP-16BIP, MP-16LF and MP-16LB and MP-16F3 and MP-16B3, the in molar ratio composition of 40:20:5.
Second object of the present invention is to provide a kind of test kit for carrying out LAMP detection to mycoplasma pneumoniae.
Test kit provided by the present invention, comprises the above-mentioned primer for carrying out LAMP detection to mycoplasma pneumoniae.
Specifically, described test kit comprises the following reagent for 25 μ L reaction systems (not containing template): (each substances content is RM: 20mMTrisHCl (pH8.8), 10mMKCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mMMgSO
4, 1.4mMdNTPs) and 12.5 μ L, 8UBstDNApolymerase1 μ L, primer add-on is respectively: 1. 50mM primer MP-16FIP0.8 μ L, makes final concentration be 40pmol; 2. 50mM primer MP-16BIP0.8 μ L, makes final concentration be 40pmol; 3. 50mM primer MP-16LF0.4 μ L, makes final concentration be 20pmol; 4. 50mM primer MP-16LB0.4 μ L, makes final concentration be 20pmol; 5. 50mM primer MP-16F30.1 μ L, makes final concentration be 5pmol; 6. 50mM primer MP-16B30.1 μ L, makes final concentration be 5pmol.
For convenience of detecting, also can comprise positive control and negative control in described test kit, described positive control is mycoplasma pneumoniae type strain genomic dna, and described negative control is not containing the LAMP amplification system of DNA, as distilled water.
Above-mentioned LAMP primer or the application of test kit in mycoplasma pneumoniae LAMP detects also belong to protection scope of the present invention.
3rd object of the present invention is to provide a kind of using method of LAMP kit of mycoplasma pneumoniae.
The method can comprise the following steps:
1) with testing sample genomic dna for template, under the guiding of above-mentioned primer, carry out LAMP amplification, LAMP reaction system is: (each substances content is: 20mMTrisHCl (pH8.8) for testing sample genomic dna 2 μ L, RM, 10mMKCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mMMgSO
4, 1.4mMdNTPs) and 12.5 μ L, 8UBstDNApolymerase1 μ L, ddH
2o6.9 μ L, primer add-on is: 1. 50mM primer MP-16FIP0.8 μ L, makes final concentration be 40pmol; 2. 50mM primer MP-16BIP0.8 μ L, makes final concentration be 40pmol; 3. 50mM primer MP-16LF0.4 μ L, makes final concentration be 20pmol; 4. 50mM primer MP-16LB0.4 μ L, makes final concentration be 20pmol; 5. 50mM primer MP-16F30.1 μ L, makes final concentration be 5pmol; 6. 50mM primer MP-16B30.1 μ L, makes final concentration be 5pmol; LAMP amplification condition is: put 60-65 DEG C of constant temperature 45min;
2) reaction carries out result judgement after terminating: in reaction solution, add fluorexon indicator, according to the colour-change judged result of reaction solution, there is mycoplasma pneumoniae (result is positive) in green expression in testing sample, there is not mycoplasma pneumoniae (result is negative) in orange expression testing sample; Or do not add fluorexon indicator and directly carry out judged result with the turbidity change of reaction solution before and after turbidimeter detection reaction, turbidity (reduced turbidity >=0.3) rises and represents in testing sample to there is mycoplasma pneumoniae (result is positive), there is not mycoplasma pneumoniae (result is negative) in the unchanged expression testing sample of turbidity.
In the above-mentioned methods, described step 1) in LAMP reaction system in be also provided with positive control and negative control, described positive control is mycoplasma pneumoniae type strain genomic dna, and described negative control is not containing the LAMP amplification system of DNA, as distilled water; Described LAMP amplification condition is preferably: put 61 DEG C of constant temperature 45min.
Described step 2) in the addition of fluorexon indicator can be 1 μ L (end reaction system is 26 μ L), containing 0.5mM fluorexon and 10mM Manganous chloride tetrahydrate.
The invention provides a kind of LAMP detection kit of mycoplasma pneumoniae and primer special thereof and using method, its principle adopts the specific conservative target sequence of LAMP technology to mycoplasma pneumoniae P1 gene to detect.
Test kit of the present invention is used to have the following advantages:
1, specificity is high: use the identification of 6 primer pair target sequences, 8 specific regions to ensure that the high degree of specificity that LAMP increases, and namely LAMP from the gene sample differing an only Nucleotide, can find out corresponding target sequence and increase.Respectively with streptococcus aureus (Streptococcusaureus), klebsiella pneumoniae (Klebsiellapneumoniae), Neisseria meningitidis (neisseriameningitides), H7N9 subtype avian influenza virus (ThebirdfluvirusofH7N9), stenotrophomonas maltophilia (StenotrophomonasMaltophilia), Listeria (Listeriamonocytogenes), hemophilus influenzae (Haemophilusinfluenzae), shigella sonnei (Shigellasonnei), Salmonellas (salmonella), diphtheria corynebacterium (corynebacteriumdiphtheriae), tubercule bacillus (MycobacteriumTuberculosis), Acinetobacter bauamnnii (Acinetobacterbaumannii), methicillin-resistant Staphylococcus (methicillin-resistantstaphylococcus), the genomic dna of escherichia coli (escherichiacoli) and mycoplasma pneumoniae is the specificity of template detection LAMP kit of the present invention and detection method, result shows, mycoplasma pneumoniae can only be detected by LAMP kit of the present invention and detection method, other bacterium can not be detected, confirm that LAMP kit of the present invention and detection method have higher specificity.
2, highly sensitive: to carry out LAMP and PCR with the mycoplasma pneumoniae genomic dna of 10 times of gradient dilutions for template and detect, the relatively sensitivity of two kinds of detection methods, result display can detect the mycoplasma pneumoniae genomic dna of 0.039ng/ μ L concentration by LAMP kit of the present invention and detection method, its sensitivity is 10 times of PCR detection method.
3, simple to operate, equipment requirements is low.Detection sample and detection reaction liquid only need be put into after 60-65 DEG C of thermostat water bath hatches 45min and get final product judged result by the present invention.
4, detection time is short, amplification efficiency is high.Whole LAMP amplified reaction can complete in one hour, and goal gene productive rate can reach 0.5mg/mL.
5, result interpretation is easy: detected result judges, without the need to complex instrument by visual inspection (fluorexon colour developing) or turbidimeter.
In sum, use test kit of the present invention can realize under isothermal conditions fast, convenient, efficient, height is special, detect mycoplasma pneumoniae with sensitivity, without the need to complex instrument, the detection for mycoplasma pneumoniae provides new technology platform.The present invention can be used for Animal husbandry production unit, primary care health unit and the examination of each disease prevention and control center and detects mycoplasma pneumoniae, has wide market outlook and larger economical, societal benefits, is suitable for applying on a large scale.
Below in conjunction with specific embodiment, the present invention is described in further details.
Accompanying drawing explanation
Fig. 1 is the LAMP response curve of five cover combination of primers;
Fig. 2 is the specific turbidimeter detected result of mycoplasma pneumoniae LAMP detection method;
Fig. 3 is mycoplasma pneumoniae LAMP detection method specific fluorexon indicator detected result;
Fig. 4 is the turbidimeter detected result of mycoplasma pneumoniae LAMP detection method sensitivity;
Fig. 5 is the fluorexon indicator detected result of mycoplasma pneumoniae LAMP detection method sensitivity;
Fig. 6 is the detected result of mycoplasma pneumoniae regular-PCR detection method sensitivity.
Embodiment
In following embodiment, method therefor is ordinary method if no special instructions, concrete steps can be see: " MolecularCloning:ALaboratoryManual " (Sambrook, J., Russell, DavidW., MolecularCloning:ALaboratoryManual, 3rdedition, 2001, NY, ColdSpringHarbor).
Described percentage concentration is mass/mass (W/W if no special instructions, unit g/100g) percentage concentration, mass/volume (W/V, unit g/100mL) percentage concentration or volume/volume (V/V, Unit/mL/100mL) percentage concentration.
The approach that obtains of the various biomaterials be described in embodiment is only to provide a kind of approach of testing acquisition to reach concrete disclosed object, should not become the restriction to biological material source of the present invention.In fact, the source of used biomaterial is widely, and any biomaterial that can obtain with moral ethics that keeps on the right side of the law can replace use according to the prompting in embodiment.
The primer is synthesized by Beijing biological company limited of raw work.
Embodiment is implemented under premised on technical solution of the present invention, gives detailed embodiment and concrete operating process, and embodiment will contribute to understanding the present invention, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1, for carrying out the design of primers of LAMP detection to mycoplasma pneumoniae
Mycoplasma pneumoniae P1 gene (No. GenBank: CP002077.1) is obtained from nucleic acid database GenBank (http://www.ncbi.nlm.nih.gov) retrieval of NCBI, homology analysis is carried out by BLAST software, obtain specific conservative's target sequence (SEQ ID NO:1) of mycoplasma pneumoniae P1 gene, again according to this conservative target sequence, primer mycoplasma pneumoniae being carried out to LAMP detection is designed for software PrimerdesignV4 (http://primerexplorer.jp/e/), (code name is MP-4 to design five cover combination of primers, MP-16, MP-22, MP-27, MP-50, through Experimental comparison, finally have chosen combination of primers MP-16, primer sequence is as shown in table 1.
Table 1 is for carrying out the combination of primers MP-16 of LAMP detection to mycoplasma pneumoniae
The LAMP of embodiment 2, mycoplasma pneumoniae detects
One, the best combination of primers that mycoplasma pneumoniae LAMP detects is determined
Five covers combination of primers (code name is MP-4, MP-16, MP-22, MP-27, MP-50) designed by embodiment 1 carry out LAMP detection to mycoplasma pneumoniae (deriving from from Diseases Preventing and Controlling Institute's infectious disease control center), to obtain best combination of primers, concrete grammar is as follows:
1) genomic dna of mycoplasma pneumoniae is extracted
With QIAampviralDNAminikit (Qiagen, Hilden, Germany) commercialization DNA extraction kit extract mycoplasma pneumoniae genomic dna, concrete extracting method comprises the following steps:
1. draw 560 μ L ready AVL damping fluid-carrierRNA (vector rna is provided by QIAampviralDNAminikit test kit) to (actual amount per sample adjusts AVL damping fluid-carrierRNA in proportion) in 1.5mL centrifuge tube.
2. 140 μ L blood plasma, serum, urine, culturing cell supernatant liquor or acellular body fluid being joined is equipped with in the centrifuge tube of bufferAVL-carrierRNA, mediates 15 seconds, mixing.
3. room temperature places 10min.
4. brief centrifugation, gets rid of the drop on lid at the bottom of return pipe.
5. add 560 μ L dehydrated alcohols (96%-100%) in sample, mediation 15s fully mixes.Then brief centrifugation, gets rid of the drop on lid at the bottom of return pipe.
6. the solution drawn on 630 μ L in step adds in column (adsorption column) (being encased in 2mL centrifuge tube) carefully, notes the edge not encountering pillar.Cover lid, 6000 × g (8000rpm) centrifugal 1min.Column is put into new 2mL centrifuge tube, discard old collection tube.
7. the careful lid opening column, repeats the 6th step.
8. careful open column lid, add 500 μ LbufferAW1.Cover lid, 8000rpm is centrifugal, 1min.Column is put into new 2mL collection tube, discard old collection tube.
9. careful open column lid, add 500 μ LbufferAW2.Cover lid, at full speed centrifugal (14000rpm) 3min.
10. column is placed in 1.5mL centrifuge tube.Discard old collection tube.Careful opens column, adds the bufferAVE of 60 μ L room temperatures.Cover lid, room temperature places 1min.8000rpm centrifugal 1min is the genomic dna of mycoplasma pneumoniae in centrifuge tube.
2) under the guiding of above-mentioned five cover combination of primers, LAMP amplification is carried out respectively, 25 μ LLAMP reaction systems comprise: the genomic dna 2 μ L of mycoplasma pneumoniae, (each substances content is RM: 20mMTrisHCl (pH8.8), 10mMKCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mMMgSO
4, 1.4mMdNTPs) and 12.5 μ L, 8UBstDNApolymerase1 μ L, ddH
2o6.9 μ L, primer add-on is respectively: 1. 50mM primer MP-16FIP0.8 μ L, makes final concentration be 40pmol; 2. 50mM primer MP-16BIP0.8 μ L, makes final concentration be 40pmol; 3. 50mM primer MP-16LF0.4 μ L, makes final concentration be 20pmol; 4. 50mM primer MP-16LB0.4 μ L, makes final concentration be 20pmol; 5. 50mM primer MP-16F30.1 μ L, makes final concentration be 5pmol; 6. 50mM primer MP-16B30.1 μ L, makes final concentration be 5pmol; LAMP amplification condition is: put 60-65 DEG C of constant temperature 45min.
3) reaction is carried out result and is judged after terminating: in reaction solution, add the fluorexon indicator of 1 μ L (end reaction system as 26 μ L) containing 0.5mM fluorexon and 10mM Manganous chloride tetrahydrate, according to colour-change judged result (principle: fluorexon is Metal ion indicator, the Mg in energy Indicator Reaction liquid of reaction solution
2+change), green represent in testing sample to there is mycoplasma pneumoniae (positive), in orange expression testing sample, there is not mycoplasma pneumoniae (feminine gender); Or do not add fluorexon indicator directly to carry out judged result with the change of the turbidity of reaction solution before and after turbidimeter detection reaction and (in the process that principle: LAMP reacts, can magnesium pyrophosphate be produced, magnesium pyrophosphate is a kind of white precipitate, according to the change of turbidity, turbidimeter can judge that LAMP reacts), turbidity rises and represents in testing sample to there is mycoplasma pneumoniae (positive), there is not mycoplasma pneumoniae (feminine gender) in the unchanged expression testing sample of turbidity.
The ring mediated isothermal nucleic acid amplification instrument of the LA230 model of company of Eiken Chemical carries out isothermal amplification and records result, (X-coordinate is the reaction times to LAMP response curve such as Fig. 1 of five cover combination of primers, ordinate zou is for reaction product is at the turbidity value of 650nm) shown in, as can be seen from the figure the reaction effect of combination of primers MP-16 is best.
Therefore, the present invention determines that MP-16 is best combination of primers, comprise: primer MP-16F3 (SEQIDNO:2) and MP-16B3 (SEQIDNO:3), primer MP-16FIP (SEQIDNO:4) and MP-16BIP (SEQIDNO:5), and primer LF (SEQIDNO:6) and LB (SEQIDNO:7).Illustrate and see embodiment 1.
Two, the optimal reaction system that mycoplasma pneumoniae LAMP detects is determined
Carry out LAMP detection with combination of primers MP-16 to mycoplasma pneumoniae (deriving from from Diseases Preventing and Controlling Institute's infectious disease control center), to obtain optimal reaction system, concrete grammar is as follows:
1) under the guiding of combination of primers MP-16, carry out LAMP amplification, 25 μ LLAMP reaction systems comprise: the genomic dna 2 μ L of mycoplasma pneumoniae, RM12.5 μ L, ddH
2o6.9 μ L, 8UBstDNApolymerase1 μ L, primer add-on is: 1. 50mM primer MP-16FIP0.8ul, makes final concentration be 40pmol; 2. the MP-16BIP0.8 μ L of 50mM, makes final concentration be 40pmol; 3. 50mM primer MP-16LF0.4 μ L, makes final concentration be 20pmol; 4. 50mM primer MP-16LB0.4 μ L, makes final concentration be 20pmol; 5. 50mM primer MP-16F30.1 μ L, makes final concentration be 5pmol; 6. 50mM primer MP-16B30.1 μ L, makes final concentration be 5pmol; LAMP amplification condition is: put 60-65 DEG C of constant temperature 45min;
Combination of primers adds scheme:
40pmolMP-16FIP and MP-16BIP, 20pmolMP-16LF and MP-16LB, 10pmolMP-16F3 and MP-16B3;
40pmolMP-16FIP and MP-16BIP, 20pmolMP-16LF and MP-16LB, 5pmolMP-16F3 and MP-16B3;
30pmolMP-16FIP and MP-16BIP, 20pmolMP-16LF and MP-16LB, 5pmolMP-16F3 and MP-16B3;
30pmolMP-16FIP and MP-16BIP, 20pmolMP-16LF and MP-16LB, 10pmolMP-16F3 and MP-16B3;
40pmolMP-16FIP and MP-16BIP, 10pmolMP-16LF and MP-16LB, 5pmolMP-16F3 and MP-16B3.
2) reaction carries out result judgement after terminating: identical with step one.
Result: combination of primers 40pmol primer MP-16FIP and MP-16BIP, 20pmol primer MP-16LF and MP-16LB, 5pmol primer MP-16F3 and MP-16B3, reaction effect is best.
The LAMP detection system of best mycoplasma pneumoniae is decided to be (25 μ L): testing sample genomic dna 2 μ L, RM12.5 μ L, ddH
2o6.9 μ L, 8UBstDNApolymerase1 μ L, primer add-on is: 1. 50mM primer MP-16FIP0.8 μ L, makes final concentration be 40pmol; 2. 50mM primer MP-16BIP0.8 μ L, makes final concentration be 40pmol; 3. 50mM primer MP-16LF0.4 μ L, makes final concentration be 20pmol; 4. 50mM primer MP-16LB0.4 μ L, makes final concentration be 20pmol; 5. 50mM primer MP-16F30.1 μ L, makes final concentration be 5pmol; 6. 50mM primer MP-16B30.1 μ L, makes final concentration be 5pmol.
Three, the best amplification condition that mycoplasma pneumoniae LAMP detects is determined
Carry out LAMP detection with combination of primers MP-16 to mycoplasma pneumoniae, to obtain best amplification condition, concrete grammar is as follows:
1) under the guiding of combination of primers MP-16, carry out LAMP amplification, 25 μ LLAMP reaction systems comprise: identical with step 2; LAMP amplification condition is: put 60-65 DEG C of constant temperature 45min;
2) reaction carries out result judgement after terminating: identical with step one.
Result is under the LAMP amplification condition of 60-65 DEG C of constant temperature 45min, and combination of primers MP-16 all achieves good reaction effect, and particularly under the LAMP amplification condition of 61 DEG C of constant temperature 45min, reaction effect is best.
The LAMP amplification condition of best mycoplasma pneumoniae is: put 60-65 DEG C of constant temperature 45min, preferably put 61 DEG C of constant temperature 45min.
The specific detection of the LAMP detection method of embodiment 3, mycoplasma pneumoniae of the present invention
Respectively with streptococcus aureus (Streptococcusaureus), klebsiella pneumoniae (Klebsiellapneumoniae), for Neisseria meningitidis (neisseriameningitides), H7N9 subtype avian influenza virus (ThebirdfluvirusofH7N9), stenotrophomonas maltophilia (StenotrophomonasMaltophilia), Listeria (Listeriamonocytogenes), hemophilus influenzae (Haemophilusinfluenzae), shigella sonnei (Shigellasonnei), Salmonellas (salmonella), diphtheria corynebacterium (corynebacteriumdiphtheriae), tubercule bacillus (MycobacteriumTuberculosis), Acinetobacter bauamnnii (Acinetobacterbaumannii), methicillin-resistant Staphylococcus (methicillin-resistantstaphylococcus), escherichia coli (escherichiacoli), the genomic dna of negative control (distilled water) and positive control (mycoplasma pneumoniae (Mp) reference culture) (above bacterial strain is all from Diseases Preventing and Controlling Institute's infectious disease control center) is testing sample, LAMP detection is carried out, to detect the specificity of LAMP primer of the present invention and LAMP detection method by the LAMP detection method in the LAMP primer composition MP-16 in embodiment 1 and embodiment 2.
Specific turbidimeter detected result (1 streptococcus aureus as shown in Figure 2 of LAMP primer of the present invention and LAMP detection method, 2 klebsiella pneumoniaes, 3 Neisseria meningitidis, 4H7N9 subtype avian influenza virus, 5 stenotrophomonas maltophilias, 6 Listerias, 7 hemophilus influenzaes, 8 shigella sonneis, 9 Salmonellass, 10 diphtheria corynebacteriums, 11 tubercule bacillus, 12 Acinetobacter bauamnniis, 13 methicillin-resistant Staphylococcus, 14 escherichia colis, 15 negative controls, 16 positive controls), as can be seen from the figure, 16 positive controls (mycoplasma pneumoniae (Mp) reference culture) are only had to there occurs LAMP reaction (turbidity rising), all there is not LAMP reaction (turbidity is unchanged) in all the other, show that LAMP detection method and the primer special thereof of mycoplasma pneumoniae of the present invention have higher specificity, mycoplasma pneumoniae can be detected specifically.
The specific fluorexon indicator detected result of LAMP primer of the present invention and LAMP detection method is as Fig. 3 (1 streptococcus aureus, 2 klebsiella pneumoniaes, 3 Neisseria meningitidis, 4H7N9 subtype avian influenza virus, 5 stenotrophomonas maltophilias, 6 Listerias, 7 hemophilus influenzaes, 8 shigella sonneis, 9 Salmonellass, 10 diphtheria corynebacteriums, 11 tubercule bacillus, 12 Acinetobacter bauamnniis, 13 methicillin-resistant Staphylococcus, 14 escherichia colis, 15 negative controls, 16 positive controls, "+" represents the result positive (green), "-" represents result feminine gender (orange)) shown in, fluorexon indicator detection method is consistent with the result of turbidimeter detection method, 16 positive controls (mycoplasma pneumoniae (Mp) reference culture) result is only had to be positive, all the other results are all negative, LAMP detection method and the primer special thereof of further proof mycoplasma pneumoniae of the present invention have higher specificity, mycoplasma pneumoniae can be detected specifically.
The sensitivity technique of the LAMP detection method of embodiment 4, mycoplasma pneumoniae of the present invention
Detect the sensitivity that LAMP method of the present invention and regular-PCR method detect mycoplasma pneumoniae, method is: the genomic dna extracting mycoplasma pneumoniae, then dilute with 10 times of gradients, again with the mycoplasma pneumoniae genomic dna through gradient dilution for template, the concentration of mycoplasma pneumoniae genomic dna in 1-7 template is made to be respectively 39ng/ μ L, 3.9ng/ μ L, 0.39ng/ μ L, 0.039ng/ μ L (39pg/ μ L), 0.0039ng/ μ L (3.9pg/ μ L), 0.00039ng/ μ L (0.39pg/ μ L), 0.000039ng/ μ L (0.039pg/ μ L), the LAMP method in the embodiment of the present invention 2 and regular-PCR (primer is MP-16F3 and MP-16B3) method is used to carry out sensitivity technique respectively.
The turbidimeter detected result of the sensitivity of LAMP method of the present invention is as shown in Figure 4 shown in (each lines corresponding templates concentration is respectively 39ng/ μ L, 3.9ng/ μ L, 0.39ng/ μ L, 0.039ng/ μ L, 0.0039ng/ μ L, 0.00039ng/ μ L, 0.000039ng/ μ L), and (template concentrations of 1-7 bottle is respectively fluorexon indicator detected result such as Fig. 5 of the sensitivity of LAMP method of the present invention: 39ng/ μ L, 3.9ng/ μ L, 0.39ng/ μ L, 0.039ng/ μ L, 0.0039ng/ μ L, 0.00039ng/ μ L, 0.000039ng/ μ L; "+" represents the result positive (green), "-" represents result feminine gender (orange)) shown in, (template concentrations of swimming lane 1-6 is by 10 times of dilution gradients for detected result such as Fig. 6 of regular-PCR, be respectively: 39ng/ μ L, 3.9ng/ μ L, 0.39ng/ μ L, 0.039ng/ μ L, 0.0039ng/ μ L, 0.00039ng/ μ L) shown in, target product clip size is 210bp.Can find out, detect with the LAMP of mycoplasma pneumoniae of the present invention and 0.039ng/ μ L mycoplasma pneumoniae genomic dna (Fig. 4 and Fig. 5 display) can be detected, and regular-PCR method only can detect the 0.39ng/ μ L mycoplasma pneumoniae genomic dna band of label 3 (in the Fig. 6), show that the sensitivity of mycoplasma pneumoniae LAMP detection method is 10 times of PCR detection method, and fluorexon indicator detection method is consistent with the result of turbidimeter detection method.
The LAMP detection kit of embodiment 5, mycoplasma pneumoniae
The LAMP detection kit of mycoplasma pneumoniae provided by the invention comprises: outer primer MP-16F3 and MP-16B3, inner primer MP-16FIP and MP-16BIP and ring primer MP-16LF and MP-16LB.Wherein the nucleotide sequence of six primers is respectively as shown in SEQ ID NO:2 (MP-16F3), SEQIDNO:3 (MP-16B3), SEQIDNO:4 (MP-16FIP), SEQIDNO:5 (MP-16BIP), SEQIDNO:6 (MP-16LF) and SEQIDNO:7 (MP-16LB).
Concrete, also can by 20mMTrisHCl (pH8.8), 10mMKCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mMMgSO
4, 1.4mMdNTPs, 8UBstDNApolymerase, primer add-on is 5pmol primer MP-16F3 and MP-16B3 (shown in SEQIDNO:2 and SEQIDNO:3 primer), 40pmol primer MP-16FIP and MP-16BIP (shown in SEQIDNO:4 and SEQIDNO:5 primer), 20pmol primer MP-16LF and MP-16LB (shown in SEQIDNO:6 and SEQIDNO:7 primer), as the mycoplasma pneumoniae type strain genomic dna of positive control, and jointly pack as the distilled water of negative control, obtain the LAMP detection kit of the mycoplasma pneumoniae for 25 μ L reaction systems.
The LAMP detection kit of this mycoplasma pneumoniae can the method for reference example 2 use.Specifically can comprise the following steps:
1) with testing sample genomic dna for template, under the guiding of described primer, carry out LAMP amplification, LAMP reaction system is: (each substances content is: 20mMTrisHCl (pH8.8) for testing sample genomic dna 2 μ L, RM, 10mMKCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mMMgSO
4, 1.4mMdNTPs) and 12.5 μ L, 8UBstDNApolymerase1 μ L, ddH
2o6.9 μ L, primer add-on is: 1. 50mM primer MP-16FIP0.8 μ L, makes final concentration be 40pmol; 2. 50mM primer MP-16BIP0.8 μ L, makes final concentration be 40pmol; 3. 50mM primer MP-16LF0.4 μ L, makes final concentration be 20pmol; 4. 50mM primer MP-16LB0.4 μ L, makes final concentration be 20pmol; 5. 50mM primer MP-16F30.1 μ L, makes final concentration be 5pmol; 6. 50mM primer MP-16B30.1 μ L, makes final concentration be 5pmol; LAMP amplification condition is: put 60-65 DEG C of constant temperature 45min;
2) reaction carries out result judgement after terminating: in reaction solution, add fluorexon indicator, according to the colour-change judged result of reaction solution, there is mycoplasma pneumoniae (result is positive) in green expression in testing sample, there is not mycoplasma pneumoniae (result is negative) in orange expression testing sample; Or do not add fluorexon indicator and directly carry out judged result with the turbidity change of reaction solution before and after turbidimeter detection reaction, turbidity (reduced turbidity >=0.3) rises and represents in testing sample to there is mycoplasma pneumoniae (result is positive), there is not mycoplasma pneumoniae (result is negative) in the unchanged expression testing sample of turbidity.
Claims (10)
1. for detecting the LAMP primer of mycoplasma pneumoniae, design according to specific conservative's target sequence of mycoplasma pneumoniae P1 gene (No. GenBank: CP002077.1), described LAMP primer is made up of six primers, comprises the combination of outer primer MP-16F3 and MP-16B3, inner primer MP-16FIP and MP-16BIP and ring primer MP-16LF and MP-16LB; Specific conservative's target sequence of described mycoplasma pneumoniae pneumonia mycoplasma P1 gene is as shown in SEQ ID NO:1.
2. the LAMP primer for detecting mycoplasma pneumoniae according to claim 1, is characterized in that: the nucleotide sequence of described six primers for carrying out LAMP detection to mycoplasma pneumoniae is as shown in SEQ ID NO:2 (MP-16F3), SEQIDNO:3 (MP-16B3), SEQIDNO:4 (MP-16FIP), SEQIDNO:5 (MP-16BIP), SEQIDNO:6 (MP-16LF) and SEQIDNO:7 (MP-16LB).
3. the LAMP primer for detecting mycoplasma pneumoniae according to claim 1 and 2, is characterized in that: described LAMP primer is the composition of MP-16FIP and MP-16BIP, MP-16LF and MP-16LB and MP-16F3 and MP-16B3 40:20:5 in molar ratio.
4., for carrying out a test kit for LAMP detection to mycoplasma pneumoniae, comprise described in claim 1 or 2 or 3 for carrying out the primer of LAMP detection to mycoplasma pneumoniae.
5. test kit according to claim 4, it is characterized in that: described test kit comprises the following reagent for 25 μ L reaction systems (not containing template): (each substances content is RM: 20mMTrisHCl (pH8.8), 10mMKCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mMMgSO
4, 1.4mMdNTPs) and 12.5 μ L, 8UBstDNApolymerase1 μ L, primer add-on is respectively: 1. 50mM primer MP-16FIP0.8 μ L, makes final concentration be 40pmol; 2. 50mM primer MP-16BIP0.8 μ L, makes final concentration be 40pmol; 3. 50mM primer MP-16LF0.4 μ L, makes final concentration be 20pmol; 4. 50mM primer MP-16LB0.4 μ L, makes final concentration be 20pmol; 5. 50mM primer MP-16F30.1 μ L, makes final concentration be 5pmol; 6. 50mM primer MP-16B30.1 μ L, makes final concentration be 5pmol.
6. the test kit according to claim 4 or 5, it is characterized in that: in described test kit, also can comprise positive control and negative control, described positive control is mycoplasma pneumoniae type strain genomic dna, and described negative control is not containing the LAMP amplification system of DNA, as distilled water.
7. claim 1 or the LAMP primer described in 2 or 3 or claim 4 or the application of the test kit described in 5 or 6 in the LAMP of mycoplasma pneumoniae detects.
8. apply according to claim 7, it is characterized in that: the LAMP for mycoplasma pneumoniae detects, and comprises the following steps:
1) with testing sample genomic dna for template, under the guiding of above-mentioned primer, carry out LAMP amplification, LAMP reaction system is: (each substances content is: 20mMTrisHCl (pH8.8) for testing sample genomic dna 2 μ L, RM, 10mMKCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mMMgSO
4, 1.4mMdNTPs) and 12.5 μ L, 8UBstDNApolymerase1 μ L, ddH
2o6.9 μ L, primer add-on is: 1. 50mM primer MP-16FIP0.8 μ L, makes final concentration be 40pmol; 2. 50mM primer MP-16BIP0.8 μ L, makes final concentration be 40pmol; 3. 50mM primer MP-16LF0.4 μ L, makes final concentration be 20pmol; 4. 50mM primer MP-16LB0.4 μ L, makes final concentration be 20pmol; 5. 50mM primer MP-16F30.1 μ L, makes final concentration be 5pmol; 6. 50mM primer MP-16B30.1 μ L, makes final concentration be 5pmol; LAMP amplification condition is: put 60-65 DEG C of constant temperature 45min;
2) reaction carries out result judgement after terminating: in reaction solution, add fluorexon indicator, according to the colour-change judged result of reaction solution, there is mycoplasma pneumoniae (result is positive) in green expression in testing sample, there is not mycoplasma pneumoniae (result is negative) in orange expression testing sample; Or do not add fluorexon indicator and directly carry out judged result with the turbidity change of reaction solution before and after turbidimeter detection reaction, turbidity (reduced turbidity >=0.3) rises and represents in testing sample to there is mycoplasma pneumoniae (result is positive), there is not mycoplasma pneumoniae (result is negative) in the unchanged expression testing sample of turbidity.
9. application according to claim 8, it is characterized in that: described step 1) in LAMP reaction system in be also provided with positive control and negative control, described positive control is mycoplasma pneumoniae type strain genomic dna, described negative control is not containing the LAMP amplification system of DNA, as distilled water; Described LAMP amplification condition is preferably: put 61 DEG C of constant temperature 45min.
10. application according to claim 8, is characterized in that: described step 2) in the addition of fluorexon indicator can be 1 μ L (end reaction system is 26 μ L), containing 0.5mM fluorexon and 10mM Manganous chloride tetrahydrate.
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