Primer group for visually detecting drug-resistant gene of MCR-3 polymyxin and detection method thereof
阅读说明：本技术 一种可视化检测mcr-3多粘菌素耐药基因的引物组及其检测方法 (Primer group for visually detecting drug-resistant gene of MCR-3 polymyxin and detection method thereof ) 是由 郝智慧 刘志海 郭长梅 黄亭亭 于 2019-09-25 设计创作，主要内容包括：本发明提供了一种可视化检测MCR-3多粘菌素耐药基因的引物组及其检测方法,属于细菌耐药基因的检测领域。其技术方案为：引物组包括外引物F3和B3,内引物FIP和BIP；在62℃对DNA样品进行LAMP扩增反应,在LAMP反应体系中预先添加适量羟基萘酚兰(HNB),通过观察反应液的颜色变化判断待测物中是否存在MCR-3基因。本发明的有益效果为：本发明能够在恒温条件下快速、高效、高特异、高灵敏地检测到MCR-3基因,无需昂贵复杂的仪器,为细菌耐药基因的检测提供了新的方法,具有较大的应用价值。(The invention provides a primer group for visually detecting an MCR-3 polymyxin drug-resistant gene and a detection method thereof, belonging to the field of detection of bacterial drug-resistant genes. The technical scheme is as follows: the primer group comprises outer primers F3 and B3, and inner primers FIP and BIP; performing LAMP amplification reaction on a DNA sample at 62 ℃, adding a proper amount of hydroxynaphthol blue (HNB) in an LAMP reaction system in advance, and judging whether the MCR-3 gene exists in the object to be detected by observing the color change of the reaction solution. The invention has the beneficial effects that: the invention can detect the MCR-3 gene rapidly, efficiently, specifically and sensitively under the constant temperature condition without expensive and complicated instruments, provides a new method for detecting the drug-resistant gene of bacteria, and has great application value.)
1. A primer group for visually detecting an MCR-3 polymyxin drug resistance gene and a detection method thereof are characterized in that the primer group comprises outer primers F3 and B3, and inner primers FIP and BIP;
the nucleotide sequences of the primers in the primer group are respectively as follows:
the outer primer F3: CAACACCATCCGCTACAC, respectively;
the outer primer B3: TCCAGGTGACATCCACAC, respectively;
the inner primer FIP: AGCAACGCGGTGTTGTACTTGATTGGAGAGATGATTGCCA, respectively;
the inner primer BIP: CATGGTGAATCACTGGGAGCATTAAGGAACACGGGTCTGATC, respectively;
the detection method comprises the following steps:
s1, extracting DNA of a sample to be detected;
s2, taking the genomic DNA extracted in the step S1 as a template, conducting isothermal LAMP amplification reaction under the guide of the primer group, and adding a proper amount of hydroxynaphthol blue (HNB) in the LAMP reaction system in advance;
s3, judging the result: according to the result of the color change judgment of the reaction solution in the step S2, sky blue indicates the presence of the MCR-3 gene in the sample to be tested, and purple indicates the absence of the MCR-3 gene in the sample to be tested.
2. The primer group for visually detecting the MCR-3 polymyxin drug-resistant gene, as claimed in claim 1, wherein the primer group further comprises DNA molecules with the same functions of the primer sequences of the outer primer F3, the outer primer B3, the inner primer FIP and the inner primer BIP after the substitution and/or deletion and/or addition of one or more nucleotides.
3. The primer group for visually detecting the MCR-3 polymyxin drug-resistant gene and the detection method thereof according to claim 2, wherein the 25ul reaction system of the LAMP amplification reaction in the step 2 comprises: 1. mu.l of genomic DNA of the test article, 0.8M betaine (betaine), 180. mu. mol/L hydroxynaphthol blue, 8mM MgSO4, 1.4mM dNTPeach, 8U BstDNA polymerase, 0.2. mu.M each of the inner primers and 1.6. mu.M each of the outer primers were made up to 25. mu.l with sterilized deionized water.
4. The detection method according to claim 2, characterized in that: the LAMP amplification reaction conditions in the step S2 are as follows: keeping the temperature at 62 ℃ for 60 min.
Technical Field
The invention relates to the field of detection of bacterial drug resistance genes, in particular to a primer group for visually detecting MCR-3 polymyxin drug resistance genes and a detection method thereof.
Background
Since the 20 th century, a large number of natural antibiotics were discovered in succession, opening the antibiotic era, but with an excessive dependence on the use of antibiotics, the problem of bacterial resistance became increasingly prominent. Resistant bacteria cause antibiotic drugs to be less effective, or even ineffective. Polymyxins (Polymyxins) are a group of polypeptide antibiotics extracted from culture fluid of polymyxa bacteria, and have 5 components (A, B, C, D, E). Polymyxin (polymyxin b) and polymyxin E (colistin, preducin) are commonly used clinically. Polymyxin is considered the last line of defense against bacteria in humans;
in recent years, the rapid evolution of plasmid-mediated polymyxin drug resistance gene MCR and its wide spread in the world pose serious threats to public health and human health, so that a new mobile polymyxin drug resistance gene MCR-3 has been detected in large quantities in multi-drug resistant intestinal bacteria carried by severe patients in multiple countries since the first discovery in shandong, china, and MCR-3 positive bacteria were discovered in 8 out of 13 nations;
due to the wide existence of MCR-3, particularly in healthy people and animal food, the detection procedures of selective bacteria screening, mass spectrum identification of bacteria, drug sensitive detection and common PCR verification for MCR detection become very complicated and inefficient. And expensive instruments and equipment are needed, and the like, and the method cannot be clinically popularized and used in a large scale.
Disclosure of Invention
The invention aims to provide a primer group for visually detecting an MCR-3 polymyxin drug-resistant gene and a detection method thereof, wherein the primer group can be used for quickly, efficiently, highly specifically and sensitively detecting the MCR-3 gene under a constant temperature condition, expensive and complex instruments are not needed, a new method is provided for detecting the bacterial drug-resistant gene, and the primer group has a high application value.
The invention is realized by the following measures:
a primer group for visually detecting an MCR-3 polymyxin drug resistance gene and a detection method thereof are characterized in that the primer group comprises outer primers F3 and B3, and inner primers FIP and BIP;
the nucleotide sequences of the primers in the primer group are respectively as follows:
the outer primer F3: CAACACCATCCGCTACAC, respectively;
the outer primer B3: TCCAGGTGACATCCACAC, respectively;
the inner primer FIP: AGCAACGCGGTGTTGTACTTGATTGGAGAGATGATTGCCA, respectively;
the inner primer BIP: CATGGTGAATCACTGGGAGCATTAAGGAACACGGGTCTGATC, respectively;
the inner Primer and the outer Primer are used for obtaining an mcr-3 gene sequence by searching from an American gene database, carrying out homology analysis by BLAST software to obtain a specific conserved target sequence of the mcr-3 gene, and designing a Primer for LAMP detection of the mcr-3 gene by using software Primer design V4 according to the conserved target DNA sequence;
the detection method comprises the following steps:
s1, extracting DNA of a sample to be detected;
s2, taking the genomic DNA extracted in the step S1 as a template, conducting isothermal LAMP amplification reaction under the guide of the primer group, and adding a proper amount of hydroxynaphthol blue (HNB) in the LAMP reaction system in advance;
s3, judging the result: according to the result of the color change judgment of the reaction solution in the step S2, sky blue indicates the presence of the MCR-3 gene in the sample to be tested, and purple indicates the absence of the MCR-3 gene in the sample to be tested.
The invention has the following specific characteristics:
the primer group also comprises DNA molecules which have the same functions with the primer sequences of the outer primer F3, the outer primer B3, the inner primer FIP and the inner primer BIP after the primer sequences are substituted and/or deleted and/or added by one or more nucleotides.
The 25ul reaction system of the LAMP amplification reaction in the
The LAMP amplification reaction conditions in the step S2 are as follows: keeping the temperature at 62 ℃ for 60 min.
The invention has the beneficial effects that: the invention can detect the MCR-3 gene rapidly, efficiently, specifically and sensitively under the constant temperature condition without expensive and complex instruments, provides a new method for detecting the drug-resistant gene of bacteria, and has great application value;
among them, high specificity: the identification of 6 specific regions of the mcr-3 gene target sequence by the 4 primers ensures the high specificity of LAMP amplification, namely LAMP can find out the corresponding target sequence from a gene sample with only one nucleotide difference for amplification;
high sensitivity: the sensitivity is 10 times higher than that of the common PCR;
the result identification is simple and convenient: the results can be observed by naked eyes (HNB color development);
the operation is simple: the result can be judged only by putting the detection sample and the detection reagent into a constant-temperature water bath kettle at 62 ℃ for 60 minutes;
rapid and efficient amplification: the whole LAMP amplification reaction can be completed within one hour.
Drawings
FIG. 1 shows the LAMP detection result in the first experiment according to the embodiment of the present invention.
FIG. 2 shows the result of LAMP detection determined by electrophoresis in the first experiment of the present invention.
FIG. 3 shows the specific detection result of the experimental triple LAMP detection method in the embodiment of the present invention.
FIG. 4 shows the specific detection result of LAMP detection determined by three experiments in the present invention.
FIG. 5(A) is the sensitivity detection result of the experimental triple LAMP detection method in the example of the present invention.
FIG. 5(B) is the sensitivity detection result of the experimental triple LAMP detection method in the example of the present invention.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
A primer group for visually detecting an MCR-3 polymyxin drug resistance gene and a detection method thereof are characterized in that the primer group comprises outer primers F3 and B3, and inner primers FIP and BIP;
the nucleotide sequences of the primers in the primer group are respectively shown as follows:
outer primer F3: CAACACCATCCGCTACAC, respectively;
outer primer B3: TCCAGGTGACATCCACAC, respectively;
inner primer FIP: AGCAACGCGGTGTTGTACTTGATTGGAGAGATGATTGCCA, respectively;
the inner primer BIP: CATGGTGAATCACTGGGAGCATTAAGGAACACGGGTCTGATC, respectively;
an inner Primer and an outer Primer, wherein the mcr-3 gene sequence is obtained by searching from the American gene database, homology analysis is carried out through BLAST software to obtain a specific conserved target sequence of the mcr-3 gene, and a Primer for LAMP detection of the mcr-3 gene is designed by using software Primer design V4 according to the conserved target DNA sequence;
the detection method comprises the following steps:
s1, extracting DNA of a sample to be detected;
s2, taking the genomic DNA extracted in the step S1 as a template, conducting isothermal LAMP amplification reaction under the guide of the primer group, adding a proper amount of hydroxynaphthol blue (HNB) in advance in an LAMP reaction system, and placing the reaction solution in a water bath heating pot;
s3, judging the result: according to the result of the color change judgment of the reaction solution in the step S2, sky blue indicates the presence of the MCR-3 gene in the sample to be tested, and purple indicates the absence of the MCR-3 gene in the sample to be tested.
The invention has the following specific characteristics:
the primer group also comprises DNA molecules which have the same functions with the sequences after the primer sequences of the outer primer F3, the outer primer B3, the inner primer FIP and the inner primer BIP are substituted and/or deleted and/or added by one or more nucleotides.
The 25ul reaction system of the LAMP amplification reaction in the
The LAMP amplification reaction conditions in step S2 are: keeping the temperature at 62 ℃ for 60 min.
The inventor of the primer group and the detection method of the invention carries out a series of experimental researches on the reaction conditions and the performances of the detection method, and determines to prepare the primer group and the detection method of the invention, so that the primer group and the detection method of the invention have obvious high-efficiency detection advantages compared with the prior art.
Experiment I, establishment of LAMP amplification reaction system in the invention
The four primers used for LAMP detection of the mcr-3 gene in example 1 were used for LAMP detection of Escherichia coli containing the mcr-3 gene to obtain the optimal reaction system and conditions, and the specific method was as follows:
determination of optimal reaction System
Adding Mg with different concentrations into the reaction system under the same reaction condition (keeping the temperature at 62 ℃ for 60min) 2+To determine an optimal reaction system, comprising the steps of:
1) LAMP amplification is carried out under the guide of four primers obtained in example 1 by taking genome DNA of escherichia coli containing mcr-3 gene as a template, wherein 25ul LAMP reaction system comprises: mu.l of genomic DNA of Escherichia coli containing mcr-3 gene, 0.8M betaine (betaine), 0.2. mu.M each of inner primer and 1.6. mu.M each of outer primer, 180. mu. mol/L hydroxynaphthol blue (HNB) added (6 mM, 7mM, 8mM, 9mM, 10mM) MgSO4, 1.4mM dNTPeach, 8UBst DNA polymerase, respectively; the reaction condition is that the reactor is placed in a water bath kettle with the constant temperature of 62 ℃ for 60 min.
2) And (3) judging the result after the reaction is finished: judging the result according to the color change of the reaction solution (principle: hydroxynaphthol blue is a metal ion indicator and can indicate Mg in the reaction solution 2+A change in (c). ) The sky blue indicates the presence of mcr-3 gene in the test sample (positive), the purple indicates the absence of mcr-3 gene in the test sample (negative), see FIG. 1, the left tube shows sky blue, and the middle and right tubes show purple.
Determination result at 8mM Mg 2+At the concentration, the reaction result is best, so the best LAMP detection system of mcr-3 gene is determined as (25 ul): 1. mu.l of genomic DNA of the test substance, 0.8M betaine (betaine), 180. mu. mol/L hydroxynaphthol blue, 8mM MgSO4, 1.4mM dNTPeach, 8U Bst DNA polymerase, and 0.2 each of the inner primersMu M and 1.6 mu M of each outer primer are filled to 25 mu l by using sterilized deionized water.
Experiment two, determination of conditions of the detection method of the present invention
Performing LAMP detection on genomic DNA of Escherichia coli containing mcr-3 gene under different reaction conditions in the same reaction system determined in experiment one to determine the optimal reaction conditions, comprising the following steps:
1) LAMP amplification was performed under the guidance of the four primers obtained in example 1 using genomic DNA of E.coli containing mcr-3 gene as a template, and reactions were performed at different constant temperatures (60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃) and reaction times (40min, 50min, 60min, 80min, 90min), respectively.
2) The result judging method is the same as the step I.
The reaction result is best when the temperature is kept at 62 ℃ for 60 min; therefore, the optimal LAMP reaction condition of the mcr-3 gene is set as constant temperature reaction at 62 ℃ for 60 min.
Experiment III, detection method specificity and sensitivity detection in the invention
Specific detection of LAMP detection method of mcr-3 gene
The specificity of the LAMP detection method for detecting the optimal MCR-3 gene obtained in example two was determined by using the genomic DNA of two strains of E.coli containing the MCR-3 gene and ten strains of E.coli containing no MCR-3 gene but NDM-5, OXA-10, TEM-1B, OXA-1, CTX-m-65, MCR-1, TET (A), sul2, sul3 and QnrD as templates and DEPC water as a negative control, and the detection results are shown in FIG. 3.
After the reaction is finished, 2% agarose gel detection is carried out on the LAMP amplification product, the detection results are shown in figure 4 (M, DNA marker D2000; 1 and 2, two strains of escherichia coli containing MCR-3 gene; 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, namely escherichia coli containing NDM-5, OXA-10, TEM-1B, OXA-1, CTX-M-65, MCR-1, TET (A), sul2, sul3 and QnrD respectively; 13, negative control (DEPC water), specific bands are detected in the escherichia coli containing MCR-3 gene, and the specific LAMP bands are not detected in the other strains without MCR-3 gene, and the result is consistent with the result of the hydroxynaphthol blue (HNB) staining method in the first step in the third experiment, which shows that the LAMP detection method of the MCR-3 gene has higher specificity, the mcr-3 gene can be specifically detected in a plurality of bacterial drug resistance genes.
Secondly, the sensitivity detection of the LAMP detection method of the mcr-3 gene
The detection method of the LAMP detection method and the sensitivity of detecting the mcr-3 gene by the common PCR method comprises the following steps: extracting total DNA of Escherichia coli containing mcr-3 gene, and gradient (1 time, 10 times) by 10
The result determination method is the same as the first step in example 2. The detection results are shown in FIG. 5 (A: LAMP sensitivity detection result, B: common PCR sensitivity detection result, 1-fold dilution, 2, 10-fold dilution, 3, 10-fold dilution) 2Carrying out dilution; 4,10 3Carrying out dilution; 5,10 4Carrying out dilution; 6,10 5Carrying out dilution; 7,10 6Carrying out dilution; 8,10 7Carrying out dilution; 9,10 8Carrying out dilution; 10, 10 9Carrying out dilution; 11, 10 10Fold dilution), the LAMP detection method of the mcr-3 gene can detect 10 2The dilution concentration is doubled, but the common PCR method can only detect 10 times of dilution concentration, which shows that the LAMP detection method of the mcr-3 gene is 10 times higher than the common PCR detection method in sensitivity.
The technical features of the present invention which are not described in the above embodiments may be implemented by or using the prior art, and are not described herein again, of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and variations, modifications, additions or substitutions which may be made by those skilled in the art within the spirit and scope of the present invention should also fall within the protection scope of the present invention.
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