CN104630354A - LAMP kit and special primer thereof for detecting streptococcus pyogenes - Google Patents
LAMP kit and special primer thereof for detecting streptococcus pyogenes Download PDFInfo
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Abstract
The invention discloses an LAMP kit and a special primer thereof for detecting streptococcus pyogenes. The LAMP primer for detecting streptococcus pyogenes is designed according to a specific conservative target sequence of a streptococcus pyogenes transcriptional modulatory gene spy1258, and comprises a combination of outer primers F3 and B3, inner primers FIP and BIP and a loop primer LF. By utilizing the LAMP kit, streptococcus pyogenes in pure strains, phlegm, cerebrospinal fluid, other secretions and other samples can be qualitatively detected quickly, conveniently, efficiently, specifically and sensitively under an isothermal condition without complex instrument, and a new technical platform is provided to detection of streptococcus pyogenes.
Description
Technical field
The invention belongs to biological technical field, relate to the molecular biology of bacterium, particularly relate to the LAMP kit of a kind of streptococcus pyogenes and primer special and its thereof and detecting the application in streptococcus pyogenes.
Background technology
Streptococcus pyogenes clinical infection and current treatment status
Streptococcus pyogenes (Streptococcus pyogenes), i.e. A group streptococcus (Group A streptococcus, GAS, be A group according to antigen construct different definition), β haemolysis (refer to and can make Sheep Blood cell agar haemolysis completely), it is one of important pathogenic bacteria of human body, a variety of disease can be caused, the infectious diseases caused mainly contains acute pharyngitis, acute tonsillitis, also can cause pulmonary infection, scarlet fever, skin soft-tissue infection, and can systemic infection be caused.Streptococcus pyogenes is also the remote cause causing allergic disorder rheumatic fever and acute glomerulonephritis.In recent years, A group streptococcus often causes serious infection, and the sickness rate of the disease caused by aggressive A group streptococcus (invasive group A streptococcus infections) increases, and people are paid close attention to more to this bacterial infection.
Streptococcus pyogenes can attack the people at any age, but a patient mostly is children.Normal people's pharynx nasalis, skin can carry disease germs, and have to be carried disease germs by anus, vagina and cause the report of outbreak of epidemic.Respiratory tract all can be propagated with directly contacting.Also the report that the contaminated food of feed causes angor to break out is had.Be poorly off, sanitary condition is poor, it is crowded to live, close contact etc. all contributes to the generation that streptococcus pyogenes infects.
At present, the first-selected penicillin of the medicine that streptococcus pyogenes infects, but should be taken into account there is Resistant strain, answers escalated dose or uses its medicine instead, as erythromycin, clindamycin, the first-generation, two generation cephalosporin analog antibiotic etc.Preferably to select with reference to local drug sensitivity tests.
Detect culture identification, serological analysis etc. that the method for streptococcus pyogenes is mainly traditional, there is the shortcomings such as consuming time and result not easily judges.Therefore, detecting pathogenic bacteria fast, accurately, give the antibiotic therapy of normative and reasonable, is the effective measure controlling streptococcus pyogenes infection.
LAMP technology is introduced
Ring mediated constant temperature nucleic acid amplification technology (1oop-mediated isothermal amplification, LAMP) be by Notomi (Notomi T, et al.Loop-mediated isothermal amplification of DNA.Nucleic Acids Res 2000; 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.People (the Imai M such as Masaki Imai, Ninomiya A, Minekawa H, et al.Rapid diagnosis of H5N1 avian influenza virus infection by newly developed influenza H5hemagglutinin gene-specific loop-mediated isothermal amplification method.J Virol Methods.2007; 141 (2): 173-80.) LAMP is utilized to establish the Laboratory Diagnosed system of avian influenza virus; People (the Hayashi N such as Nobuyuki Hayashi, Arai R, Tada S, Taguchi H, Ogawa Y.Detection and identification of Brettanomyces/Dekkera sp.yeasts with a loop-mediated isothermal amplification method.Food Microbiol.2007; 24 (7-8): 778-85.) devise LAMP Auele Specific Primer for the ITS sequence of four kinds of cordiale yeast, establish efficient LAMP detection system.LAMP can also 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 (work HHNV), tomato spotted wilf virus, tomato yellow leaf curl virus etc.At present, the LAMP kit for detecting streptococcus pyogenes and primer special thereof is showed no both at home and abroad.
Summary of the invention
The invention provides the primer for carrying out LAMP detection to streptococcus pyogenes, to realize the batch detection of streptococcus pyogenes, improve the specificity and sensitivity that detect.
LAMP primer for detecting streptococcus pyogenes provided by the present invention, design according to specific conservative's target sequence of streptococcus pyogenes transcriptional modulatory gene (the putative transcriptional regulator gene) spy1258, in order to the streptococcus pyogenes in the samples such as the pure bacterium of qualitative detection, sputum, cerebrospinal fluid and other secretory product, described LAMP primer is made up of five primers, comprises the combination of outer primer F3 and B3, inner primer FIP and BIP and ring primer LF; Specific conservative's target sequence of described streptococcus pyogenes transcriptional modulatory gene spy1258 is as shown in SEQ ID NO:1 in sequence table.
Specifically, the nucleotide sequence of described five primers for carrying out LAMP detection to streptococcus pyogenes is as shown in SEQ ID NO:2 (F3), SEQ ID NO:3 (B3), SEQ ID NO:4 (FIP), SEQ ID NO:5 (BIP) and SEQ ID NO:6 (LF) in sequence table.
Described LAMP primer is the composition of FIP and BIP, F3 and B3 and LF 40:5:20 in molar ratio.
Second object of the present invention is to provide a kind of test kit for carrying out LAMP detection to streptococcus pyogenes.
Test kit provided by the present invention, comprises the above-mentioned primer for carrying out LAMP detection to streptococcus pyogenes.
Specifically, described test kit comprises the following reagent for 25 μ l reaction systems: 20mM TrisHCl (pH8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTP each, 8U Bst DNA polymerase, primer add-on is: 40pmol primers F IP and BIP, 5pmol primers F 3 and B3,20pmol primer LF.
For convenience of detecting, also can comprise positive control and negative control in described test kit, described positive control is streptococcus pyogenes 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 streptococcus pyogenes LAMP detects also belong to protection scope of the present invention.
3rd object of the present invention is to provide the LAMP detection method of a kind of streptococcus pyogenes.
Detection method provided by the present invention, can comprise the following steps:
1) with testing sample genomic dna for template, under the guiding of above-mentioned primer, carry out LAMP amplification, 25 μ l LAMP reaction systems comprise: testing sample genomic dna 2 μ l, 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTP each, 8U Bst DNA polymerase, primer add-on is: 40pmol primers F IP and BIP, 5pmol primers F 3 and B3,20pmol primer LF; LAMP amplification condition is: put 60-65 DEG C of constant temperature 45-55min;
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 streptococcus pyogenes, there is not streptococcus pyogenes in orange expression testing sample in green 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 rises and represents in testing sample to there is streptococcus pyogenes, there is not streptococcus pyogenes in the unchanged expression testing sample of turbidity.
In above-mentioned detection method, described step 1) in LAMP reaction system in be also provided with positive control and negative control, described positive control is streptococcus pyogenes 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 63 DEG C of constant temperature 50min.
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 the LAMP detection method of a kind of streptococcus pyogenes and primer special thereof and test kit.The streptococcus pyogenes principle that the present invention detects adopts LAMP technology, detects specific conservative's target sequence of streptococcus pyogenes transcriptional modulatory gene spy1258.Spy1258 is the natural transcriptional modulatory gene carried of streptococcus pyogenes, can be used as one of streptococcus pyogenes marker gene.
Test kit of the present invention is used to have the following advantages:
1, 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 45-55min and get final product judged result by the present invention.Detected result judges, without the need to complex instrument by visual inspection (fluorexon colour developing) or turbidimeter.
2, specificity is good, highly sensitive.The identification of five primer pair streptococcus pyogenes target sequence specific regions of the present invention's design ensure that the high degree of specificity that LAMP increase, and namely LAMP can find out corresponding target sequence and increases from the gene sample differing only 1 Nucleotide; Sensitivity comparable regular-PCR height 10-100 doubly.
3, 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.
In sum, use test kit of the present invention can realize under isothermal conditions fast, convenient, efficient, height is special, detect streptococcus pyogenes with sensitivity, without the need to complex instrument, the detection for streptococcus pyogenes 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 streptococcus pyogenes, 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 three cover primers
Fig. 2 is the specific turbidimeter detected result of streptococcus pyogenes LAMP detection method
Fig. 3 is streptococcus pyogenes LAMP detection method specific fluorexon indicator detected result
Fig. 4 is the turbidimeter detected result of streptococcus pyogenes LAMP detection method sensitivity
Fig. 5 is the fluorexon indicator detected result of streptococcus pyogenes LAMP detection method sensitivity
Fig. 6 is the detected result of streptococcus pyogenes regular-PCR detection method sensitivity
Embodiment
In following embodiment, method therefor is ordinary method if no special instructions, concrete steps can be see: " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).
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 synthesizes by Beijing Sheng Gong biotech firm.
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 streptococcus pyogenes
Streptococcus pyogenes transcriptional modulatory gene spy1258 sequence (No. GenBank: AE006565) 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 (in sequence table SEQ ID NO:1) of streptococcus pyogenes transcriptional modulatory gene spy1258, again according to this conservative target sequence, primer streptococcus pyogenes being carried out to LAMP detection is designed for software Primer design V4, (code name is 36 to design three cover combination of primers, 84 and 106), through Experimental comparison, finally have chosen combination of primers 106, primer sequence is as shown in table 1.
Table 1 is for carrying out the combination of primers 106 of LAMP detection to streptococcus pyogenes
The LAMP of embodiment 2, streptococcus pyogenes detects
One, the best combination of primers that streptococcus pyogenes LAMP detects is determined
Three covers combination of primers (code name 36,84 and 106) designed by embodiment 1 carry out LAMP detection to streptococcus pyogenes (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) under the guiding of above-mentioned three cover combination of primers, carry out LAMP amplification respectively, 25 μ l LAMP reaction systems comprise: streptococcus pyogenes genomic dna (Promega
genomic DNA Purification Kit A1125 extracts the nucleic acid in testing sample) 2 μ l, 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTP each, 8U Bst DNA polymerase (purchased from NEB company), primer add-on is: 40pmol primers F IP and BIP, 5pmol primers F 3 and B3,20pmol primer LF; LAMP amplification condition is: put 63 DEG C of constant temperature 50min;
2) 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 streptococcus pyogenes (positive), in orange expression testing sample, there is not streptococcus pyogenes (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 streptococcus pyogenes (positive), streptococcus pyogenes (feminine gender) is there is not in the unchanged expression testing sample of turbidity, (X-coordinate is the reaction times to LAMP response curve such as Fig. 1 of three 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 106 is best.
Therefore, the present invention is best, and combination of primers is: primers F 3 (SEQ ID NO:2) and B3 (SEQ ID NO:3), primers F IP (SEQ ID NO:4) and BIP (SEQ ID NO:5), and primer LF (SEQ ID NO:6).
Two, the optimal reaction system that streptococcus pyogenes LAMP detects is determined
LAMP detection is carried out with combination of primers 106 pairs of streptococcus pyogeness (deriving from from Diseases Preventing and Controlling Institute's infectious disease control center), the conventional LAMP of reference laboratory tests reaction system and determines that the LAMP detection system (25 μ l) of the streptococcus pyogenes carrying out LAMP amplification under the guiding of combination of primers 106 is: testing sample genomic dna 2 μ l, 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTP each, 8U Bst DNA polymerase, primer add-on is: 40pmol primers F IP and BIP, 5pmol primers F 3 and B3,20pmol primer LF.
Three, the best amplification condition that streptococcus pyogenes LAMP detects is determined
Carry out LAMP detection with combination of primers 106 pairs of streptococcus pyogeness, to obtain best amplification condition, concrete grammar is as follows:
1) under the guiding of combination of primers 106, carry out LAMP amplification, 25 μ l LAMP reaction systems comprise: identical with step one; LAMP amplification condition is: put 60-65 DEG C of constant temperature 45-55min;
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 45-55min, and combination of primers 106 all achieves good reaction effect, and particularly under the LAMP amplification condition of 63 DEG C of constant temperature 50min, reaction effect is best.
The LAMP amplification condition of best streptococcus pyogenes is: put 60-65 DEG C of constant temperature 45-55min, preferably put 63 DEG C of constant temperature 50min.
Specificity, the susceptibility of the LAMP detection method of embodiment 3, streptococcus pyogenes detect
One, the specific detection of the LAMP detection method of streptococcus pyogenes
Respectively with Acinetobacter baumannii, acinetobacter lwoffii, the special bacterium of Whooping cough Boulder, corynebacterium diphtheriae, enteroinvasive E.Coli, enteropathogenic Escherichia coli, enterotoxigenic E.Coli, Klebsiella Pneumoniae, mycobacterium tuberculosis, Neisseria meningitidis A group, Neisseria meningitidis B group, Neisseria meningitidis C group, Neisseria meningitidis, Pseudomonas aeruginosa, salmonella enteritis serotype Paratyphi, Salmonella enteritidis, shigella flexneri, Song Shi shigella, stenotrophomonas maltophilia, streptococcus pneumoniae, streptococcus pneumoniae, shark vibrio, vibrio cholerae, Vibrio parahemolyticus, Yersinia enterocolitica, yersinia pestis, the genomic dna of streptococcus pyogenes (above bacterial strain is all from Diseases Preventing and Controlling Institute's infectious disease control center) is template, take distilled water as negative control, detect the specificity of the LAMP detection scheme of the streptococcus pyogenes that embodiment 2 obtains.
Turbidimeter detected result is (1 Acinetobacter baumannii B260 as shown in Figure 2, 2 Acinetobacter baumannii F398, 3 Acinetobacter baumannii H18, 4 Acinetobacter baumannii H949, 5 acinetobacter lwoffii JN49-1, the special bacterium ATCC 18530 of 6 Whooping cough Boulders, 7 corynebacterium diphtheriae CMCC 38001, 8 enteroinvasive E.Colis 44825, 9 enteropathogenic Escherichia colis 2348, 10 enterotoxigenic E.Colis 44824, 11 Klebsiella Pneumoniaes 46117, 12 mycobacterium tuberculosis 4368, 13 Neisseria meningitidis A group CMCC 29202, 14 Neisseria meningitidis B group B CMCC 29022, 15 Neisseria meningitidis C group CMCC29026, 16 Neisseria meningitidis Y group CMCC 29028, 17 Pseudomonas aeruginosa P104, 18 salmonella enteritis serotype Paratyphi 86423, 19 Salmonella enteritidis 50326-1, 20 shigella flexneris 4536, 21 Song Shi shigellas 2531, 22 stenotrophomonas maltophilia s17, 23 streptococcus pneumoniae CMCC31001, 24 streptococcus pneumoniae SP112-07, 25 shark vibrios 5732, 26 cholera vibrio O 139s, 27 Vibrio parahemolyticus 5474, 28 Yersinia enterocolitica Pa3606, 29 yersinia pestis 201, 30 negative controls, 31 streptococcus pyogeness), as can be seen from the figure, 31 (streptococcus pyogeness) are only had to there occurs LAMP reaction (turbidity rising), all there is not LAMP reaction (turbidity is unchanged) in all the other, fluorexon indicator detected result is as Fig. 3 (1 Acinetobacter baumannii B260, 2 Acinetobacter baumannii F398, 3 Acinetobacter baumannii H18, 4 Acinetobacter baumannii H949, 5 acinetobacter lwoffii JN49-1, the special bacterium ATCC 18530 of 6 Whooping cough Boulders, 7 corynebacterium diphtheriae CMCC38001, 8 enteroinvasive E.Colis 44825, 9 enteropathogenic Escherichia colis 2348, 10 enterotoxigenic E.Colis 44824, 11 Klebsiella Pneumoniaes 46117, 12 mycobacterium tuberculosis 4368, 13 Neisseria meningitidis A group CMCC 29202, 14 Neisseria meningitidis B group B CMCC 29022, 15 Neisseria meningitidis C group CMCC29026, 16 Neisseria meningitidis Y group CMCC 29028, 17 Pseudomonas aeruginosa P104, 18 salmonella enteritis serotype Paratyphi 86423, 19 Salmonella enteritidis 50326-1, 20 shigella flexneris 4536, 21 Song Shi shigellas 2531, 22 stenotrophomonas maltophilia s17, 23 streptococcus pneumoniae CMCC 31001, 24 streptococcus pneumoniae SP112-07, 25 shark vibrios 5732, 26 cholera vibrio O 139s, 27 Vibrio parahemolyticus 5474, 28 Yersinia enterocolitica Pa3606, 29 yersinia pestis 201, 30 negative controls, 31 streptococcus pyogeness, "+" 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, 31 (streptococcus pyogenes) result is only had to be positive, all the other results are all negative, show that the LAMP detection of streptococcus pyogenes has higher specificity, streptococcus pyogenes can be detected specifically.
Two, the sensitivity technique of the LAMP detection of streptococcus pyogenes
Detect LAMP and detect the sensitivity detecting streptococcus pyogenes with regular-PCR, method is: extract streptococcus pyogenes STb gene, then with 10 times of gradients (1 times, 10 times, 10
2doubly, 10
3doubly, 10
4doubly, 10
5doubly, 10
6doubly, 10
7doubly) dilute, again with the streptococcus pyogenes DNA through gradient dilution for template, the concentration of streptococcus pyogenes genomic dna in 1-8 template is made to be respectively 14.86ng/ μ l, 1.486ng/ μ l, 148.6pg/ μ l, 14.86pg/ μ l, 1.486pg/ μ l, 0.1486pg/ μ l, 0.01486pg/ μ l, 0.001486pg/ μ l, take distilled water as negative control, carry out sensitivity technique with LAMP detection and regular-PCR (primer sequence is F3 and B3) respectively.
As shown in Figure 4, (template concentrations of 1-8 is respectively fluorexon indicator detected result such as Fig. 5 of streptococcus pyogenes LAMP detection sensitivity the specific turbidimeter detected result of streptococcus pyogenes LAMP detection method: 14.86ng/ μ l, 1.486ng/ μ l, 148.6pg/ μ l, 14.86pg/ μ l, 1.486pg/ μ l, 0.1486pg/ μ l, 0.01486pg/ μ l, 0.001486pg/ μ l; "+" represents the result positive (green), "-" represents result feminine gender (orange)) shown in, the detected result of regular-PCR is as shown in Fig. 6 (template concentrations of swimming lane 1-8 is respectively: 14.86ng/ μ l, 1.486ng/ μ l, 148.6pg/ μ l, 14.86pg/ μ l, 1.486pg/ μ l, 0.1486pg/ μ l, 0.01486pg/ μ l, 0.001486pg/ μ l), target product clip size is 407bp, can find out, detect with the LAMP of streptococcus pyogenes of the present invention and 1.486pg/ μ l streptococcus pyogenes genomic dna (10 can be detected
4times weaker concn), and regular-PCR method only can detect 14.86pg/ μ l streptococcus pyogenes genomic dna (10
3times weaker concn), and fluorexon indicator detection method is consistent with the result of turbidimeter detection method, shows that the LAMP of streptococcus pyogenes detects highly sensitive 10 times that detect than regular-PCR.
Embodiment 4, the test kit detected for the LAMP of streptococcus pyogenes
By 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
41.4mM dNTP each, 8U Bst DNA polymerase, primer add-on is 5pmol primers F 3 and B3 (shown in SEQ ID NO:2 and SEQ ID NO:3 primer), 40pmol primers F IP and BIP (shown in SEQ ID NO:4 and SEQ ID NO:5 primer), 20pmol primer LF (shown in SEQ ID NO:6 primer), as the streptococcus pyogenes type strain genomic dna of positive control, and jointly pack as the distilled water of negative control, obtain the test kit detected for the LAMP of 25 μ l reaction system streptococcus pyogeness.
Claims (10)
1. for detecting the LAMP primer of streptococcus pyogenes, design according to specific conservative's target sequence of streptococcus pyogenes transcriptional modulatory gene spy1258, in order to the streptococcus pyogenes in the samples such as the pure bacterium of qualitative detection, sputum, cerebrospinal fluid and other secretory product, described LAMP primer is made up of five primers, comprises the combination of outer primer F3 and B3, inner primer FIP and BIP and ring primer LF; Specific conservative's target sequence of described streptococcus pyogenes transcriptional modulatory gene spy1258 is as shown in SEQ ID NO:1 in sequence table.
2. LAMP primer according to claim 1, is characterized in that: the nucleotide sequence of described five primers for carrying out LAMP detection to streptococcus pyogenes is as shown in SEQ ID NO:2 (F3), SEQ ID NO:3 (B3), SEQ ID NO:4 (FIP), SEQ ID NO:5 (BIP) and SEQ ID NO:6 (LF) in sequence table.
3. LAMP primer according to claim 2, is characterized in that: described LAMP primer, is the composition of F3 and B3, FIP and BIP and LF 5:40:20 in molar ratio.
4., for carrying out a test kit for LAMP detection to streptococcus pyogenes, comprise described in claim 1 or 2 or 3 for carrying out the primer of LAMP detection to streptococcus pyogenes.
5. test kit according to claim 4, is characterized in that: described test kit comprises the following reagent for 25 μ l reaction systems: 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTP each, 8U Bst DNA polymerase, primer add-on is: 40pmol primers F IP and BIP, 5pmol primers F 3 and B3,20pmol primer LF.
6. test kit according to claim 5, it is characterized in that: in described test kit, also can comprise positive control and negative control, described positive control is streptococcus pyogenes 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 streptococcus pyogenes detects.
8. apply according to claim 7, the LAMP for streptococcus pyogenes detects, and comprises the following steps:
1) with testing sample genomic dna for template, under the guiding of described primer, carry out LAMP amplification, 25 μ l LAMP reaction systems comprise: testing sample genomic dna 2 μ l, 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2sO
4, 0.1%Tween20,0.8M trimethyl-glycine (betaine), 8mM MgSO
4, 1.4mM dNTP each, 8U Bst DNA polymerase, primer add-on is: 40pmol primers F IP and BIP, 5pmol primers F 3 and B3,20pmol primer LF; LAMP amplification condition is: put 60-65 DEG C of constant temperature 45-55min;
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 streptococcus pyogenes, there is not streptococcus pyogenes in orange expression testing sample in green 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 rises and represents in testing sample to there is streptococcus pyogenes, there is not streptococcus pyogenes 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 streptococcus pyogenes 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 63 DEG C of constant temperature 50min.
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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| CN107541509A (en) * | 2016-06-29 | 2018-01-05 | 博奥生物集团有限公司 | For detecting the LAMP primer composition and its application of 6 kinds of infectious agents of mastitis for milk cows |
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| CN107541509B (en) * | 2016-06-29 | 2020-10-09 | 博奥生物集团有限公司 | LAMP primer combination for detecting 6 infectious pathogens of cow mastitis and application thereof |
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| CN108220460B (en) * | 2017-12-07 | 2021-08-03 | 广东产品质量监督检验研究院 | Food-borne streptococcus pyogenes LAMP primer group, kit and application |
| CN111534610A (en) * | 2019-11-12 | 2020-08-14 | 广州微芯生物科技有限公司 | Fluorescent quantitative PCR method for detecting toxin-producing streptococcus pyogenes and corresponding kit |
| CN115029453A (en) * | 2021-11-16 | 2022-09-09 | 江汉大学 | MNP (protein-protein) marker site of streptococcus pyogenes, primer composition, kit and application of MNP marker site |
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