CN108300769A - The quadruple fluorescence quantifying PCR method of germ oligotrophy unit cell and sul1, sul2, sul3 drug resistant gene is detected simultaneously - Google Patents
The quadruple fluorescence quantifying PCR method of germ oligotrophy unit cell and sul1, sul2, sul3 drug resistant gene is detected simultaneously Download PDFInfo
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Abstract
The invention discloses the quadruple fluorescence quantifying PCR methods for detecting germ oligotrophy unit cell and sul1, sul2, sul3 drug resistant gene simultaneously.Multiplex PCR of the present invention can save loading time, primary to be loaded.Save experimental cost, it is only necessary to a reaction tube, a reaction system.The present invention is to sul1, sul2, sul3 and ssrA standard concentration 10~106There are good linear relationship, minimum detection limit to reach 10copies/ μ L in copies/ μ L, there is good sensitivity.The present invention carries out Quadruple- PCR detections with into using four pairs of primers, and substance needs each gene sensitive special when evaluating, and when multiple evaluation needs each primer amplification not AF panel between each other；Requirement to melting temperature is especially high, needs significantly to distinguish 4 kinds of products simultaneously.
Description
Technical field
The invention belongs to field of nucleic acid detection, be related to melting curve method and meanwhile detect germ oligotrophy unit cell and
The quadruple fluorescence quantifying PCR method of sul1, sul2, sul3 drug resistant gene.
Background technology
Hospital infection rate obviously increases in recent years, and pathogenic bacteria are largely gram-Negative bacillus, Carbapenems antibiosis
Element is its last line of defense.Germ oligotrophy unit cell is important nosocomial infection bacterium, and separation rate is in non-zymocyte, only
Inferior to pseudomonas aeruginosa and Acinetobacter baumannii, illustrate germ oligotrophy unit cell inside-hospital infection important function.
Germ oligotrophy unit cell belongs to multi-drug resistant bacteria, to beta-lactam (including Carbapenems) and aminoglycoside antimicrobial
Object natural drug resistance, while drug resistance all be can express out to quinolones, macrolides and some disinfectants.It is usually used in controlling at present
The preferred antibiotic for treating Stenotrophomonas maltophilia is sulfa drugs, and carries sul1, sul2, sul3 drug resistant gene
Bacterial strain is often to sulfa drugs drug resistance, so we are to tri- kinds of drug resistant genes of germ oligotrophy unit cell and sul1, sul2, sul3
It detects simultaneously.Having more documents at present proves sul series and the drug resistant relationship of sulfa drugs Compound New Nomin.
There is presently no good methods can be resistance to germ oligotrophy unit cell and tri- kinds of sul1, sul2, sul3 simultaneously
Medicine gene is detected.
Invention content
Above-mentioned in order to solve the problems, such as, present invention research finds to carry out quadruple fluorescent quantitation using melting curve method
PCR can quickly detect tri- kinds of Sulfonamides-resistant genes of germ oligotrophy unit cell and sul1, sul2, sul3 simultaneously.
The purpose of the present invention is to provide detect germ oligotrophy unit cell and sul1, sul2, sul3 drug resistant gene simultaneously
Quadruple fluorescence quantifying PCR method.
The technical solution used in the present invention is：
The PCR primer group of germ oligotrophy unit cell and sul1, sul2, sul3 drug resistant gene is detected simultaneously, primer sets
Nucleotide sequence is as follows：
sul1-F：CGCGACACCGAGACCAAT(SEQ ID NO：1)
sul1-R：CGCTCTATCCCGATATTGCT(SEQ ID NO：2)
sul2-F：GTGGCCTATCTCAATGATATTCG(SEQ ID NO：3)
sul2-R：CCCGTCTTGCACCGAATG(SEQ ID NO：4)
sul3-F：CTGAAGTGGGCGTTGTGG(SEQ ID NO：5)
sul3-R：ATTCGCTGAACGGAGTGC(SEQ ID NO：6)
ssrA-F：TCAGTCCGGCACTAGAACAC(SEQ ID NO：7)
ssrA-R：CGAAGGCACTCCATCCC(SEQ ID NO：8).
The quadruple quantitative fluorescent PCR reagent of germ oligotrophy unit cell and sul1, sul2, sul3 drug resistant gene is detected simultaneously
Box, the kit contain primer described above.
Further, mentioned reagent box also contains Ex Taq enzymes, dNTP Mixture, Mg2+, Tli RNaseH, SYBR
Green I。
When detect germ oligotrophy unit cell and sul1, sul2, sul3 drug resistant gene quadruple fluorescence quantifying PCR method packet
Include following steps：
1) DNA of the thalline in measuring samples is extracted；
2) quadruple quantitative fluorescent PCR：Using the DNA of extraction as template, it is fixed to carry out quadruple fluorescence using primer described above
Measure PCR；
3) interpretation of result：Melting curve analysis is carried out to amplified production, is control with positive quality control product, determining in sample is
It is no containing in germ oligotrophy unit cell and thalline whether contain sul1, sul2, sul3 drug resistant gene.
Further, the quadruple quantitative fluorescent PCR reaction system in step 2) is：
Further, the quadruple quantitative fluorescent PCR response procedures in step 2) are：：95 DEG C of 10min, 95 DEG C of 10s, 58 DEG C
15s, 72 DEG C of 20s, 45 cycles.
Further, the melting curve analysis program in step 3) is：95 DEG C of denaturation 1min；65 DEG C to 99 DEG C with 0.11
DEG C/rate of s, 5 time/DEG C continuous acquisition fluorescence signals carry out melting curve analysis.
The beneficial effects of the invention are as follows：
(1) multiplex PCR of the present invention can save loading time, primary to be loaded.Save experimental cost, it is only necessary to a reaction
Pipe, a reaction system.
(2) it compared to other multiplex PCRs, such as common multiplex PCR, is needed through agarose gel electrophoresis after amplification,
Product length difference is differentiated；It needs to uncap after reaction, be easy to cause pollution.Such as sonde method multiplex PCR, it need to design and close simultaneously
At primer and probe, the modification group somewhat expensive of taqman probes, 3 sequences need to be added in one gene of detection, be easy mutual
Between generate interference.And multiple fluorescence quantitative PCR melting curve method of the present invention is very economical, fast and effectively method, entirely
Reaction process is totally-enclosed, stablizes.
(3) present invention carries out Quadruple- PCR detection with into using four pairs of primers, and substance needs the primer of each pair of design when evaluating
Will be sensitive special, when multiple evaluation, needs each primer amplification not AF panel between each other；Requirement to melting temperature is especially
Height needs significantly to distinguish 4 kinds of products simultaneously.
Description of the drawings
Fig. 1 is the expanding effect that agarose gel electrophoresis detects each pair of primer after regular-PCR；M:DL2000DNA
marker；1:sul1；2:sul2；3:sul3；4:ssrA；
Fig. 2 is the expanding effect that substance PCR detects each primer pair, and melting temperature can distinguish 4 kinds of amplified productions；Horizontal seat
It is designated as temperature, ordinate indicates fluorescence intensity；
Fig. 3 be 11 kinds of Coexistence Situations of inventive method pair testing result, A~K figure respectively indicate simultaneously detect sul1 with
Sul2, sul1 and sul3, sul1 and ssrA, sul2 and sul3, sul2 and ssrA, sul3 and ssrA, sul1, sul2 and sul3,
Sul1, sul2 and ssrA, sul2, sul3 and ssrA, sul1, sul3 and ssrA, the result of sul1, sul2, sul3 and ssrA；
Abscissa is temperature, and ordinate indicates fluorescence intensity；
Fig. 4 is the standard curve that the method for the present invention detects sul1, sul2, sul3 and ssrA respectively, and figure A~D is indicated respectively
The standard curve of sul1, sul2, sul3, ssrA；
Fig. 5 is the melting peakss curve of sul1, sul2, sul3 and ssrA that the method for the present invention detects various concentration respectively, schemes A
~D indicates the melting peakss curve of sul1, sul2, sul3, ssrA respectively；
Fig. 6 is the testing result of partial clinical sample；
The testing result of Fig. 7 samples is to carry the germ oligotrophy unit cell of sul2, and the sample standard deviation of various concentration can be had
Effect detection.
Specific implementation mode
With reference to specific embodiment, the present invention is further illustrated.
Embodiment 1
The present invention according to the nucleic acid sequence and Sulfonamides-resistant genes sul1, sul2 of germ oligotrophy unit cell kind,
Sul3 genes, the quadruple fluorescence designed for detecting germ oligotrophy unit cell and sul1, sul2, sul3 drug resistant gene simultaneously are fixed
Measure PCR primer；It is high and special to filter out one group of sensitivity by largely being screened to designed primer and probe by the present invention
Anisotropic strong primer sequence, sequence are as follows：
sul1-F：CGCGACACCGAGACCAAT(SEQ ID NO：1)
sul1-R：CGCTCTATCCCGATATTGCT(SEQ ID NO：2)
sul2-F：GTGGCCTATCTCAATGATATTCG(SEQ ID NO：3)
sul2-R：CCCGTCTTGCACCGAATG(SEQ ID NO：4)
sul3-F：CTGAAGTGGGCGTTGTGG(SEQ ID NO：5)
sul3-R：ATTCGCTGAACGGAGTGC(SEQ ID NO：6)
ssrA-F：TCAGTCCGGCACTAGAACAC(SEQ ID NO：7)
ssrA-R：CGAAGGCACTCCATCCC(SEQ ID NO：8).
2 regular-PCR of embodiment detects the expanding effect of each primer
Method：
Positive quality control product：To contain the plasmid through correctly each target gene sequence is sequenced.
Using positive quality control product as template, respectively with primer sul1-F and sul1-R, sul2-F and sul2- shown in embodiment 1
R, sul3-F and sul3-R, ssrA-F and ssrA-R carry out regular-PCR amplification, and pcr amplification product is examined through agarose gel electrophoresis
Survey expanding effect.
The specific reaction system of regular-PCR：10.0 μ l of Premix Ex Taq, upstream and downstream are sequentially added in 0.2ml PCR pipes
Each 0.8 μ l of primer, 1 μ l of DNA profiling supply 20 μ l with aqua sterilisa.Response procedures：95 DEG C of 5min, 94 DEG C of 30s, 58 DEG C of 30s, 72
DEG C 1min, 30 cycles；72℃10min.PCR product is detected with 1% agarose gel electrophoresis confirms that purpose band has amplification.
As a result：The testing result of regular-PCR amplification is special as shown in Figure 1, it can be seen that each band expanding effect is apparent
Anisotropic good, the size of gained amplified fragments meets expection.
3 substance PCR of embodiment detects the expanding effect of each primer
Method：
Reaction system：10.0 μ l of SYBR Premix Ex Taq are sequentially added in 0.2ml PCR pipes, upstream and downstream primer is each
0.8 μ l, 1 μ l of DNA profiling (positive quality control product) supply 20 μ l with aqua sterilisa.Response procedures：95 DEG C of 10min, 95 DEG C of 10 s, 58
DEG C 15s, 72 DEG C of 20s, 45 cycles；65-99 DEG C of drafting melting curve.
As a result：Testing result as shown in Fig. 2, there it can be seen that it can be seen that substance PCR amplification is with obvious effects,
Specificity is good, and melting temperature can distinguish each amplified production；Characteristic peak when they coexist is simulated, shows each characteristic peak interval
More than 2 DEG C, the specific solution temperature corresponding to the melting peakss of each gene is respectively：Sul1 is 93.1 DEG C, and sul2 is 85.2 DEG C,
Sul3 is 81.8 DEG C, and ssrA is 87.4 DEG C；Each amplified production can be distinguished very well (see Fig. 2).
4 multiplex PCR system of embodiment is verified
Method：
Reaction system：SYBR Premix Ex Taq 10.0 μ l, sul2, sul3 or more are sequentially added in 0.2ml PCR pipes
Swim each each 0.1 μ l of 0.5 μ l, sul1, ssrA upstream and downstream primer of primer.1 μ l of positive DNA profiling supply 20 μ l with aqua sterilisa.
Response procedures：95 DEG C of 10min, 95 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 20s, 45 cycles；It draws for 65-99 DEG C and melts song
Line.
As a result：Testing result is as shown in figure 3, common mode intends 11 kinds of Coexistence Situations, and A~K figures are indicated while being detected respectively in Fig. 3
Sul1 and sul2, sul1 and sul3, sul1 and ssrA, sul2 and sul3, sul2 and ssrA, sul3 and ssrA, sul1, sul2
And sul3, sul1, sul2 and ssrA, sul2, sul3 and ssrA, sul1, sul3 and ssrA, sul1, sul2, sul3 and ssrA
The testing result of this 11 kinds of situations.From the testing result in Fig. 3 as can be seen that various gene Coexistence Situations can preferably from
Whether there is or not peak and the detection of corresponding melting temperature is played, illustrate that all situations can be achieved effective differentiation detection in the method for the present invention.
The range of linearity of substance is verified under 5 multiple system of embodiment
Method：By positive quality control product, 10 times of gradient dilutions are done, form 106、105、104、103、102, 10copies/ μ l it is dense
Gradient is spent, pcr amplification reaction and melting curve analysis are carried out by the method described in above-described embodiment 4.
As a result：Experimental result is as shown in Figure 4 and Figure 5, and single target gene is separately added into multiple system and establishes detection
As a result the melting peakss (Fig. 5) of standard curve (Fig. 4) and corresponding amplification curve show the present invention to sul1, sul2, sul3 and ssrA
Standard concentration is 10~106There are good linear relationship, related coefficient to be all higher than 0.99 in copies/ μ L, minimum detection limit reaches
10copies/μL。
6 clinical sample of embodiment detects
Method：
1) each 20 plants of germ oligotrophy unit cell sensitive to Compound New Nomin and drug resistant is collected, extracts thallus DNA respectively.
2) quadruple quantitative fluorescent PCR：Using the DNA of extraction as template, quadruple fluorescence is carried out using primer described in embodiment 1
Quantitative PCR；
Quadruple quantitative fluorescent PCR reaction system is：
Quadruple quantitative fluorescent PCR response procedures are：95 DEG C of 10min, 95 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 20s, 45 are followed
Ring.
3) interpretation of result：Melting curve analysis is carried out to amplified production, is control with positive quality control product, determining in sample is
It is no containing in germ oligotrophy unit cell and thalline whether contain sul1, sul2, sul3 drug resistant gene.
As a result：This experiment has detected 40 plants of clinical samples altogether, and the testing result of part sample is as shown in fig. 6, Quadruple- PCR
Testing result it is consistent with the result that regular-PCR detects：The wherein 20 plants germ oligotrophy unit cells sensitive to Compound New Nomin
In, 2 plants of carrying sul1, remaining is without carrying.Other 20 plants in the drug resistant germ oligotrophy unit cell of Compound New Nomin, 17
Strain carries sul1, and 3 plants of carryings sul2,2 plants of carrying sul3 illustrate that the method for the present invention has 100% accuracy.
7 analog sample of embodiment detects
Method：Analog sample (bacterium solution is added in blood specimen, directly DNA is extracted from blood specimen with paramagnetic particle method), does 10
Times gradient dilution forms 106、105、104、103、102, 10copies/ μ l concentration gradients, by the method described in above-described embodiment 4
It carries out pcr amplification reaction and melting curve analysis, melting curve peak type figure is as shown in Figure 7.
As a result：Testing result is as shown in fig. 7, the result of this sample is to carry the germ oligotrophy unit cell of sul2, no
With capable of detecting for concentration.
8 repeatability detection of embodiment
Method：Under multiplex PCR system, to positive quality control product repeat detection three times, calculate three times the average value of CT values and
Standard deviation.
As a result：Testing result shows that batch interior repeatability is good, and the standard deviation for repeating experiment ct values three times is equal<1.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
SEQUENCE LISTING
<110>Hospital of Southern Medical University
<120>The quadruple fluorescent quantitation P of germ oligotrophy unit cell and sul1, sul2, sul3 drug resistant gene is detected simultaneously
CR methods
<130>
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>Artificial primer
<400> 1
cgcgacaccg agaccaat 18
<210> 2
<211> 20
<212> DNA
<213>Artificial primer
<400> 2
cgctctatcc cgatattgct 20
<210> 3
<211> 23
<212> DNA
<213>Artificial primer
<400> 3
gtggcctatc tcaatgatat tcg 23
<210> 4
<211> 18
<212> DNA
<213>Artificial primer
<400> 4
cccgtcttgc accgaatg 18
<210> 5
<211> 18
<212> DNA
<213>Artificial primer
<400> 5
ctgaagtggg cgttgtgg 18
<210> 6
<211> 18
<212> DNA
<213>Artificial primer
<400> 6
attcgctgaa cggagtgc 18
<210> 7
<211> 20
<212> DNA
<213>Artificial primer
<400> 7
tcagtccggc actagaacac 20
<210> 8
<211> 17
<212> DNA
<213>Artificial primer
<400> 8
cgaaggcact ccatccc 17
Claims (7)
1. detecting the PCR primer group of germ oligotrophy unit cell and sul1, sul2, sul3 drug resistant gene, the core of primer sets simultaneously
Nucleotide sequence is as follows：
sul1-F：CGCGACACCGAGACCAAT(SEQ ID NO：1)
sul1-R：CGCTCTATCCCGATATTGCT(SEQ ID NO：2)
sul2-F：GTGGCCTATCTCAATGATATTCG(SEQ ID NO：3)
sul2-R：CCCGTCTTGCACCGAATG(SEQ ID NO：4)
sul3-F：CTGAAGTGGGCGTTGTGG(SEQ ID NO：5)
sul3-R：ATTCGCTGAACGGAGTGC(SEQ ID NO：6)
ssrA-F：TCAGTCCGGCACTAGAACAC(SEQ ID NO：7)
ssrA-R：CGAAGGCACTCCATCCC(SEQ ID NO：8).
2. detecting the quadruple quantitative fluorescent PCR reagent of germ oligotrophy unit cell and sul1, sul2, sul3 drug resistant gene simultaneously
Box, which is characterized in that the kit contains primer described in claim 1.
3. kit according to claim 2, it is characterised in that：The kit also contains Ex Taq enzymes, dNTP
Mixture, Mg2+, Tli RNaseH, SYBR Green I.
4. when detect germ oligotrophy unit cell and sul1, sul2, sul3 drug resistant gene quadruple fluorescence quantifying PCR method,
It is characterized in that, includes the following steps：
1) DNA of the thalline in measuring samples is extracted；
2) quadruple quantitative fluorescent PCR：Using the DNA of extraction as template, it is fixed to carry out quadruple fluorescence using primer described in claim 1
Measure PCR；
3) interpretation of result：Melting curve analysis is carried out to amplified production, is control with positive quality control product, determines in sample whether contain
Have in germ oligotrophy unit cell and thalline and whether contains sul1, sul2, sul3 drug resistant gene.
5. according to the method described in claim 4, it is characterized in that：Quadruple quantitative fluorescent PCR reaction system in step 2) is：
6. according to the method described in claim 4, it is characterized in that：Quadruple quantitative fluorescent PCR response procedures in step 2)
For：：95 DEG C of 10min, 95 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 20s, 45 cycles.
7. according to the method described in claim 4, it is characterized in that：Melting curve analysis program in step 3) is：95 DEG C of changes
Property 1min；65 DEG C to 99 DEG C of rates with 0.11 DEG C/s, 5 time/DEG C continuous acquisition fluorescence signals carry out melting curve analysis.
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| CN112011631A (en) * | 2020-09-16 | 2020-12-01 | 山东省兽药质量检验所（山东省畜产品质量检测中心） | RPA detection primer group, kit and method for drug-resistant gene sul2 |
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