CN111876509A - PCR method for detecting high-toxicity acinetobacter baumannii - Google Patents
PCR method for detecting high-toxicity acinetobacter baumannii Download PDFInfo
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Abstract
The invention discloses a PCR method for detecting high-toxicity Acinetobacter baumannii, which comprises the five steps of manufacturing a primer template, manufacturing a PCR Buffer reaction system, manufacturing a 25 mu L system, and performing reaction and result judgment. The invention detects four genes of abaR, CsuA, bap and epsA by one-time PCR, adopts electrophoresis and staining methods to judge results, and has the advantages that the DNA sequence is determined in 392 bp: 480 bp: 668 bp: 1021bp obvious products show that the corresponding genes are positive, so that the toxicity of the acinetobacter baumannii is judged. The method has the advantages of simple and easy process, high detection efficiency and capability of effectively distinguishing virulence differences among strains.
Description
Technical Field
The invention relates to the technical field of detecting high-toxicity acinetobacter baumannii, in particular to a PCR method for detecting high-toxicity acinetobacter baumannii.
Background
Acinetobacter baumannii belongs to non-fermented gram-negative bacillus, widely exists in the nature, belongs to conditional pathogenic bacteria, is an important pathogenic bacterium of hospital infection, mainly causes respiratory tract infection, and can also cause bacteremia, urinary system infection, secondary meningitis, operation site infection, ventilator-associated pneumonia and the like. The difference of toxicity exists among various strains of the acinetobacter baumannii, and the infection of the strains with high toxicity often leads to high mortality of patients, and the acinetobacter baumannii has serious drug resistance and difficult treatment. Currently, only the existence of acinetobacter baumannii can be clinically identified through biochemical reaction or mass spectrometry technology, and the virulence difference among various strains cannot be effectively distinguished. ST457 is a high-toxicity strain found in China, and researches show that abaR, CsuA, entE, bap and epsA are related to toxicity of the abaR, CsuA, entE, bap and epsA, so that the toxicity can be judged by detecting abaR, CsuA, entE, bap and epsA genes.
Disclosure of Invention
In view of the above disadvantages, the present invention provides a PCR method for detecting high-virulence acinetobacter baumannii, which determines the virulence level by detecting abaR, CsuA, bap, epsA genes.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a PCR method for detecting high-toxicity Acinetobacter baumannii comprises the steps of detecting 4 genes by one-time PCR, wherein the genes are abaR, CsuA, bap and epsA respectively, the primer sequences of the abaR are F: GGCTCGACACGGTTCA, W: ATGCTGGTCTATACAAAGGG, and the primer sequence of the CsuA is F: CATCATGGGCTGCTTG, W: ATCGTGGTTCGGCATTT, the primer sequence of the bap is F: TGCTCAATGGCGGTGGT, W: GTAGTGAACAAGTGTCGGAAAGG, the primer sequence of epsA is F: GGCTTGCACCCGATTGTA, W: GAAGAGCGTTACGCAACAT, the concrete steps are as follows:
s1: primer template
Reacting for 5 minutes at the temperature of 95-100 ℃ by adopting a conventional thermal cracking method to obtain a primer template;
s2: reaction system:
PCR Buffer containing 50mM KCl, 10mM Tris-HCl and 2.5mM MgCl2A solution having a pH of 8.3 to 8.5;
s3: 25 μ L system:
the final concentration of dNTPs was adjusted to 200. mu.l, 1. mu.l of each of F, R primers 1 to 4, 4. mu.l of the Tap enzyme, and 1. mu.l of the DNA template were taken, and the remaining volume was made up to 25. mu.l with sterile water.
S4: reaction of
S41: pre-denaturing the 25 mu L system at 94 ℃ for 5min to change the double DNA chains into single DNA chains;
s42: denaturation at 94 ℃ for 30s, annealing of each primer for 30s, extension at 72 ℃ and cycling to allow hybridization to the primer template;
s43: fully extending for 10 minutes at 72 ℃ to prolong the DNA chain;
s44: storing at 4 deg.C;
s5: determination of results
And (4) judging results by adopting an electrophoresis method and a dyeing method.
Preferably, the pyrolysis temperature in the step S1 is 100 ℃.
Preferably, the annealing temperature of each primer in the step S42 is 50 ℃.
Preferably, the extension in step S42 is performed at 72 ℃ for 1min for 30 cycles.
Preferably, in step S5, at 392 bp: 480 bp: 668 bp: the obvious product of 1021bp indicates the corresponding gene is positive.
The invention has the beneficial effects that: the invention detects four genes of abaR, CsuA, bap and epsA by one-time PCR, and the method mainly comprises the steps of preparing a primer template, preparing a PCR Buffer reaction system, preparing a 25 mu L system, reacting to obtain a sample, and then judging the result by adopting an electrophoresis and dyeing method, wherein the primer template is prepared by the following steps of: 480 bp: 668 bp: 1021bp obvious products show that the corresponding genes are positive, so that the toxicity of the acinetobacter baumannii is judged. The method has the advantages of simple and easy process, high detection efficiency and capability of effectively distinguishing virulence differences among strains.
Detailed Description
The present invention will be described in detail with reference to the following embodiments for explaining the technical contents, structural features, objects and effects of the invention in detail, and it is apparent that the embodiments are a part of the embodiments of the invention, not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
adopting a multiplex PCR technology, detecting 4 genes by one-time PCR, wherein the 4 genes are respectively as follows: abaR, CsuA, bap, epsA, wherein:
1、abaR
the primer sequence is as follows: GGCTCGACACGGTTCA
W:ATGCTGGTCTATACAAAGGG
The size of the product is as follows: 1021bp
2、bap
The primer sequence is as follows: TGCTCAATGGCGGTGGT
W:GTAGTGAACAAGTGTCGGAAAGG
The size of the product is as follows: 668p
3、CsuA
The primer sequence is as follows: CATCATGGGCTGCTTG
W:ATCGTGGTTCGGCATTT
The size of the product is as follows: 490bp
4、epsA
The primer sequence is as follows: f: GGCTTGCACCCGATTGTA
W：GAAGAGCGTTACGCAACAT
The size of the product is as follows: 392bp
The specific detection process is as follows:
s1: primer template
And (2) reacting for 5 minutes at the temperature of 95-100 ℃ by adopting a conventional thermal cracking method to obtain a primer template, optimally reacting at the temperature of 100 ℃, designing the primer to take multiple PCR into consideration, and being simple and easy in the whole process.
S2: preparation of the reaction System
PCR Buffer containing 50mM KCl, 10mM Tris-HCl and 2.5mM MgCl2The pH of the solution is 8.3-8.5.
S3: preparation of 25. mu.L system
The final concentration of dNTPs was adjusted to 200. mu.l, 1. mu.l of each of F, R primers 1 to 4, 4. mu.l of the Tap enzyme, and 1. mu.l of the DNA template were taken, and the remaining volume was made up to 25. mu.l with sterile water.
S4: reaction of
S41: pre-denaturing the 25 mu L system at 94 ℃ for 5min to change the double DNA chains into single DNA chains;
s42: denaturation at 94 ℃ for 30s, annealing of each primer for 30s, extension at 72 ℃ for 1min, and cycling for 30 times to allow hybridization with the primer template;
s43: fully extending for 10 minutes at 72 ℃ to prolong the DNA chain;
s44: stored at 4 ℃.
Wherein the primer annealing temperature is optimally 20 ℃.
S5: determination of results
The results were determined by electrophoresis and staining, and the ratio of 392 bp: 480 bp: 668 bp: the obvious product of 1021bp shows that the corresponding gene is positive, thereby detecting the virulence of each strain.
Variations and modifications to the above-described embodiments may occur to those skilled in the art, which fall within the scope and spirit of the above description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. In addition, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation, the use of the term being generic or similar to other means being encompassed by the invention.
Sequence listing
<110> Guangzhou medical university affiliated first hospital (Guangzhou respiratory center)
<120> PCR method for detecting high-toxicity acinetobacter baumannii
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>7249
<212>DNA
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>1
abactcccaa accatgaaaa tatgaataga aatctcaatg agccaatcag aggccagcaa 60
agtccatctc attgtcccaa gcggatatac atccgacaaa actcaacctc acaagtagac 120
tctcccttct tccgtaacta ttcgtccacg tgtccttccc tctcaccact taccttcaaa 180
accaacgctt cttttttagt tccttggtcc gaagcgttta ccgatgagag aatcataaac 240
tcccacttgg agctcaaaaa gtgtaagaga caaccaacaa aaaacgattc atctcttctc 300
ctatcctctc ctcttcgaat tcaacgtttg gagaatccag cagccgcaaa atggcttcgc 360
ttgtgtattc tccattcact ctatccactt ctaaagcaga gcatctctct tcgctcacta 420
acagtaccaa acattctttc ctccggaaga aacacagatc aaccaaacca gccaaatctt 480
tcttcaaggt gaaatctgct gtatctggaa acggcctctt cacacagacg aacccggagg 540
tccgtcgtat agttccgatc aagagagaca acgttccgac ggtgaaaatc gtctacgtcg 600
tcctcgaggc tcagtaccag tcttctctca gtgaagccgt gcaatctctc aacaagactt 660
cgagattcgc atcctacgaa gtggttggat acttggtcga ggagcttaga gacaagaaca 720
cttacaacaa cttctgcgaa gaccttaaag acgccaacat cttcattggt tctctgatct 780
tcgtcgagga attggcgatt aaagttaagg atgcggtgga gaaggagaga gacaggatgg 840
acgcagttct tgtcttccct tcaatgcctg aggtaatgag actgaacaag cttggatctt 900
ttagtatgtc tcaattgggt cagtcaaagt ctccgttttt ccaactcttc aagaggaaga 960
aacaaggctc tgctggtttt gccgatagta tgttgaagct tgttaggact ttgcctaagg 1020
ttttgaagta cttacctagt gacaaggctc aagatgctcg tctctacatc ttgagtttac 1080
agttttggct tggaggctct cctgataatc ttcagaattt tgttaagatg atttctggat 1140
cttatgttcc ggctttgaaa ggtgtcaaaa tcgagtattc ggatccggtt ttgttcttgg 1200
atactggaat ttggcatcca cttgctccaa ccatgtacga tgatgtgaag gagtactgga 1260
actggtatga cactagaagg gacaccaatg actcactcaa gaggaaagat gcaacggttg 1320
tcggtttagt cttgcagagg agtcacattg tgactggtga tgatagtcac tatgtggctg 1380
ttatcatgga gcttgaggct agaggtgcta aggtcgttcc tatattcgca ggagggttgg 1440
atttctctgg tccagtagag aaatatttcg tagacccggt gtcgaaacag cccatcgtaa 1500
actctgctgt ctccttgact ggttttgctc ttgttggtgg acctgcaagg caggatcatc 1560
ccagggctat cgaagccctg aaaaagctcg atgttcctta ccttgtggca gtaccactgg 1620
tgttccagac gacagaggaa tggctaaaca gcacacttgg tctgcatccc atccaggtgg 1680
ctctgcaggt tgccctccct gagcttgatg gagcgatgga gccaatcgtt ttcgctggtc 1740
gtgaccctag aacaggttag tccacttatg accatattgc atttttcatg attgtcatta 1800
cgcaagagtg ggaatgtttt cttatggagg tggtttacca attgctgcag ggaagtcaca 1860
tgctctccac aagagagtgg agcaactctg catcagagcg attcgatggg gtgagctcaa 1920
aagaaaaact aaggtattgt attaccactc atccttagtg aaatcacatt ccttcatgac 1980
tttctcaaga atgtaatgta tgttctcatg aatcaactct ggctcattgc aggcagagaa 2040
gaagctggca atcactgttt tcagtttccc acctgataaa ggtaatgtag ggactgcagc 2100
ttacctcaat gtgtttgctt ccatcttctc ggtgttaaga gacctcaaga gagatggcta 2160
caatgttgaa ggccttcctg agaatgcaga gactcttatt gaagaaatca ttcatgacaa 2220
ggaggctcag ttcagcagcc ctaacctcaa tgtagcttac aaaatgggag tccgtgagta 2280
ccaagacctc actccttatg caaatgccct ggaagaaaac tgggggaaac ctccggggaa 2340
ccttaactca gatggagaga accttcttgt ctatggaaaa gcgtacggta atgttttcat 2400
cggagtgcaa ccaacatttg ggtatgaagg tgatcccatg aggctgcttt tctccaagtc 2460
agcaagtcct catcacggtt ttgctgctta ctactcttat gtagaaaaga tcttcaaagc 2520
tgatgctgtt cttcattttg gaacacatgg ttctctcgag tttatgcccg ggaagcaagt 2580
gggaatgagt gatgcttgtt ttcccgacag tcttatcggg aacattccca atgtctacta 2640
ttatgcagct aacaatccct ctgaagctac cattgcaaag aggagaagtt atgccaacac 2700
catcagttat ttgactcctc cagctgagaa tgctggtcta tacaaagggc tgaagcagtt 2760
gagtgagctg atatcgtcct atcagtctct gaaggacacg gggagaggtc cacagatcgt 2820
cagttccatc atcagcacag ctaagcaatg taatcttgat aaggatgtgg atcttccaga 2880
tgaaggcttg gagttgtcac ctaaagacag agattctgtg gttgggaaag tttattccaa 2940
gattatggag attgaatcaa ggcttttgcc gtgcgggctt cacgtcattg gagagcctcc 3000
atccgccatg gaagctgtgg ccacactggt caacattgct gctctagatc gtccggagga 3060
tgagatttca gctcttcctt ctatattagc tgagtgtgtt ggaagggaga tagaggatgt 3120
ttacagagga agcgacaagg gtatcttgag cgatgtagag cttctcaaag agatcactga 3180
tgcctcacgt ggcgctgtttccgcctttgt ggaaaaaaca acaaatagca aaggacaggt 3240
ggtggatgtg tctgacaagc ttacctcgct tcttgggttt ggaatcaatg agccatgggt 3300
tgagtatttg tccaacacca agttctacag ggcgaacaga gataagctca gaacagtgtt 3360
tggtttcctt ggagagtgcc tgaagttggt ggtcatggac aacgaactag ggagtctaat 3420
gcaagctttg gaaggcaagt acgtcgagcc tggccccgga ggtgatccca tcagaaaccc 3480
aaaggtctta ccaaccggta aaaacatcca tgccttagat cctcaggcta ttcccacaac 3540
agcagcaatg gcaagtgcca agattgtggt tgagaggttg gtagagagac agaagctcga 3600
aaacgaaggg aaatatcccg agacaatcgc gcttgttctt tggggaactg acaacatcaa 3660
aacatatggg gagtctcttg ggcaggttct ttggatgatt ggtgtgagac caattgctga 3720
tacttttgga agagtgaacc gtgtcgagcc tgtgagctta gaagaactag gaaggccgag 3780
gatcgatgta gttgttaact gctcaggggt cttccgtgat ctctttatca accaggtaac 3840
cagataaaag ctttctctct tctttcattt agattcttgt tggttggtct ctcatggtgt 3900
ttcttccttg aattatcttg acagatgaac cttcttgacc gagctatcaa gatggtggcg 3960
gagctagatg agcctgtaga gcaaaatttt gtaaggaaac acgcgttgga acaagcagag 4020
gcgcttggca ttgatattag agaggcagcg acaagagttt tctcaaacgc ttcagggtca 4080
tactcagcca acatcagtct tgctgttgaa aactcgtcat ggaacgatga gaaacagctt 4140
caggacatgt acttgagccg caaatcgttt gcgtttgata gtgatgctcc tggagcagga 4200
atggctgaga agaagcaggt ctttgagatg gctcttagca ctgcagaagt caccttccag 4260
aacctggatt cttcagagat ttctttgact gatgtgagcc actacttcga ttctgaccct 4320
acaaatctag ttcagagttt gaggaaggat aagaagaaac caagctctta cattgctgac 4380
actacaactg caaacgcgca ggtgagtgaa ttttctttgt agttcatata tgggatcagt 4440
tttaccttgc aagaacacat tccttaaggt atttgttgaa tctggcaggt gaggacacta 4500
tctgagacag tgaggctgga cgcaagaaca aagctgctga atccaaagtg gtacgaagga 4560
atgatgtcaa gtggatatga aggagttcgt gagatagaga agagactgtc caacactgtg 4620
ggatggagtg caacgtcagg tcaagtagac aattgggtct acgaggaggc caactcaact 4680
ttcatccaag acgaggagat gctgaaccgt ctcatgaaca ccaatcccaa ctccttcagg 4740
aaaatgcttc agactttctt ggaggccaat ggtcgtggct actgggacac ttccgctgaa 4800
aacatagaga agctcaagga attgtactcg caggtggaag acaagatcga agggatcgat 4860
cgataaacaa tgggatataa gcctttcttc ttgtaaatga acttaaaact ttatttttgt 4920
gtattacagt attctaccaa acgacatcat ctctctgtga ataagagaat gttttggctg 4980
acattttctg actttatatg aatgttgtgg agcttcttct ttcatctcca tttcttatat 5040
catattcata tgtgtggatt ttatatgcga agaaaatgaa actatagtta agtgtaaaca 5100
aactatggtt gagtcatgct tttgcgbatt gaagcacatt cgctgcgcag ccctcgagtc 5160
gtgaagatcg gaaaatcaat agaaatccgt agtgaacaag tgtcggaaag gccaaagaca 5220
atacctatat atatatatcc gaaatattca gaaaattcga aaaatgctga atatggaaag 5280
cgcgggtgtc agtgctgcga tggcgggtct gagcaaatcg ctcaccacgc ccttctccat 5340
caacgatatc ctaacgcgca gcaatccgga aacacgtcgc atgtccagcg tggactccga 5400
accggaaccg gaaaagctga aaccctcctc cgatcgcgag cgatcgatct ccaagtcgcc 5460
gccactctgc tgccgagatc tcggcctcta caagctgacc caacccaagg agatccaacc 5520
aagtgccagg caacccagca actatctgca gtattatgcg gcggcgatgg acaacaataa 5580
ccaccatcac caggcaacgg gcacatcgaa ctccagtgcc gccgactaca tgcagcgcaa 5640
attggcctat tttggatcca ccctcgctgc tcctttggac atgagacgct gcaccagcaa 5700
cgattccggt aagtaacctg cacgaaatta acgccattca ggctctaatg gactctgaaa 5760
agaacgctac ttattcattg gccttttgta taggatgtat gctaactttt ggtaattttc 5820
cctttacaga ctgcgactca ccaccgccat tgagcagttc cccctcggag tcgccgctat 5880
cccacgacgg cagtggattg agccgcaaga agcggtcgcg tgccgccttc agccacgccc 5940
aggtcttcga gttggagcgc cgctttgccc aacagcgcta cttgtccggt ccggaacgca 6000
gcgagatggc caagagcctg cgcctgacgg agacccaggt gaagatctgg ttccaaaacc 6060
gccgctacaa gaccaagcgc aagcagatcc agcagcacga ggccgccctt ttgggtgcca 6120
gcaagagggt tcccgtccaa gtcttggtgc gagaggatgg cagcaccacc tacgctcaca 6180
tggctgctcc cggtgctgga cacggcctcg atcccgccct gatcaacatc taccgccatc 6240
agctgcagct ggcctacggc ggacttccgc tgccacagat gcagatgcca ttcccgtact 6300
tctacccgca acacaaggtg ccgcaaccca taccgccacc aacccagagt tccagctttg 6360
tgactgcatc ctcggcgtcc tcatcgccag ttcccatccc catcccgggt gcagtgcgtc 6420
cccagcgaac gccgtgtccc agtcccaatg gccagatgat gagcgtcgag agcggagcgg 6480
agagcgttca ctcggcggcg gaggatgttg acgagaatgt ggagatcgac taggttggga 6540
gtgaccatgt ctcggaggag aaccggggct acgtttttgt gatagccgta gaaattgtgt 6600
tttagcctct atgtgtaacc aatagatgtc ccatagcttt agtagcatta agttgtagtc 6660
gtatctaccc gagagctaaa taaaattatg aattaaatsa tgatattcaa tcgtggttcg 6720
gcatttataa tttcttattt tttaatttct ttagtaaatg cgggtgaaat tggagctaaa 6780
ttaactagtc aaattgaatt attgccttct tgttctgtta ataataatgt tgtagaaaat 6840
aatgcagcaa atttaaattt tggaactata gattttggtg aagctaccac atcttttaaa 6900
ggggttttag aagctagttt agttaataat ggtaattcag gttttcagat cgagtgtgct 6960
ggtatttcaa ctgtaaaaat aatatttgga gcaggaaata atgatagtaa tattccagct 7020
tcattttcac aaaattatta tcatgcttta agtaatggta gagattttat tgcttataac 7080
ttgctctatg gtttaaataa acaagtcatt aaagcaaatg aagcttttat tcttaatgat 7140
atgaataata aaaagaatat cgatattttt ggtcaagcag cccatgatgg tagtcgtata 7200
tctaagggtg aatataaaga tatagtacca attacgattg agttttaas 7249
Claims (5)
1. A PCR method for detecting high-toxicity Acinetobacter baumannii is characterized in that: 4 genes are detected by primary PCR, wherein the genes are abaR, CsuA, bap and epsA respectively, the primer sequences of the abaR are F: GGCTCGACACGGTTCA and W: ATGCTGGTCTATACAAAGGG, and the primer sequence of the CsuA is F: CATCATGGGCTGCTTG, W: ATCGTGGTTCGGCATTT, the primer sequence of the bap is F: TGCTCAATGGCGGTGGT, W: GTAGTGAACAAGTGTCGGAAAGG, the primer sequence of epsA is F: GGCTTGCACCCGATTGTA, W: GAAGAGCGTTACGCAACAT, the concrete steps are as follows:
s1: primer template
Reacting for 5 minutes at the temperature of 95-100 ℃ by adopting a conventional thermal cracking method to obtain a primer template;
s2: reaction system:
PCR Buffer containing 50mM KCl, 10mM Tris-HCl and 2.5mM MgCl2A solution having a pH of 8.3 to 8.5;
s3: 25 μ L system:
the final concentration of dNTPs was adjusted to 200. mu.l, 1. mu.l of each of F, R primers 1 to 4, 4. mu.l of the Tap enzyme, and 1. mu.l of the DNA template were taken, and the remaining volume was made up to 25. mu.l with sterile water.
S4: reaction of
S41: pre-denaturing the 25 mu L system at 94 ℃ for 5min to change the double DNA chains into single DNA chains;
s42: denaturation at 94 ℃ for 30s, annealing of each primer for 30s, extension at 72 ℃ and cycling to allow hybridization to the primer template;
s43: fully extending for 10 minutes at 72 ℃ to prolong the DNA chain;
s44: storing at 4 deg.C;
s5: determination of results
And (4) judging results by adopting an electrophoresis method and a dyeing method.
2. The PCR method for detecting Acinetobacter baumannii with high toxicity according to claim 1, wherein the PCR method comprises the following steps: the pyrolysis temperature in the step S1 is 100 ℃.
3. The PCR method for detecting Acinetobacter baumannii with high toxicity according to claim 1, wherein the PCR method comprises the following steps: the annealing temperature of each primer in the step S42 was 50 ℃.
4. The PCR method for detecting Acinetobacter baumannii with high toxicity according to claim 1, wherein the PCR method comprises the following steps: the extension in step S42 was performed at 72 ℃ for 1min for 30 cycles.
5. The PCR method for detecting Acinetobacter baumannii with high toxicity according to claim 1, wherein the PCR method comprises the following steps: at 392bp in said step S5: 480 bp: 668 bp: the obvious product of 1021bp indicates the corresponding gene is positive.
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