CN105624297B - Detection method of plant microorganism drug resistance gene - Google Patents
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Abstract
The invention discloses a method for detecting a drug resistance gene of a plant microorganism. The method comprises the following steps: determining a drug resistance gene, an endogenous standard gene and an exogenous standard gene; preparing a multiplex amplification primer; adding exogenous nucleic acid into a sample to be detected to obtain a mixed sample and extracting a genome; adding exogenous standard genes into a genome to obtain mixed nucleic acid; amplifying the mixed nucleic acid, and constructing a high-throughput sequencing library by using the amplification product and performing high-throughput sequencing to obtain a sequencing fragment group; analyzing the sequencing fragment group, and judging whether the experiment is successful according to the obtained number of sequencing fragments of the exogenous standard gene and the endogenous standard gene; if the experiment is successful, calculating the content of the drug-resistant gene; and judging whether the sample to be detected contains the drug resistance gene or not according to the content of the drug resistance gene. The method can quantitatively detect any multiple drug resistance genes to be detected in any microorganisms at one time, does not need to culture and purify the microorganisms in the detection, and has high speed and accurate and reliable result.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for detecting a drug resistance gene of a plant microorganism.
Background
The antibiotic drugs are widely applied to prevention and treatment of plant diseases, and the phenomenon of abuse of the antibiotic drugs is very common in the prevention and treatment process of the plant diseases at present, so that pathogenic microorganisms are led to evolve drug resistance genes to generate drug resistance, and the antibiotic drugs are enabled to lose efficacy in prevention and treatment of the plant diseases. Therefore, it is necessary to specifically administer the drug according to the drug resistance gene of the microorganism in the plant to avoid the drug resistance of pathogenic bacteria, and to reduce the amount of the pesticide used and the cost.
The existing methods for detecting drug-resistant genes include: estimating pathogenic bacteria of drug-resistant genes to be detected carried on a sample to be detected, separating and purifying the pathogenic bacteria, amplifying the drug-resistant genes by using a PCR (polymerase Chain Reaction) primer, and judging whether the drug-resistant genes to be detected exist in the sample to be detected one by using an electrophoresis or real-time quantitative PCR method.
In the process of implementing the invention, the inventor finds that the prior art has at least one of the following problems:
pathogenic bacteria need to be cultured, separated and purified, so that the detection time is longer, and the treatment time is delayed; the drug resistance genes in the non-culturable drug-resistant bacteria cannot be detected; only one or a few pathogenic bacteria can be detected at a time; only one or a few drug resistance genes can be detected at one time, all the drug resistance genes in a sample to be detected cannot be detected, and comprehensive information required by medication cannot be given; quantitative detection of drug resistance genes cannot be achieved.
Disclosure of Invention
In order to solve the problem that a method for detecting a drug-resistant gene in the prior art needs to be improved, the embodiment of the invention provides a method for detecting a drug-resistant gene of a plant microorganism. The technical scheme is as follows:
the embodiment of the invention provides a method for detecting a drug resistance gene of a plant microorganism, which comprises the following steps:
determining a drug resistance gene of a microorganism to be detected in a sample to be detected, an endogenous standard gene in the sample to be detected and an exogenous standard gene of the sample to be detected, wherein the sample to be detected is a whole plant or a part of the plant;
preparing multiplex amplification primers for amplifying test regions of the drug resistance gene, the endogenous standard gene and the exogenous standard gene;
extracting the genome of the sample to be detected;
adding the exogenous standard gene into the genome of the sample to be detected to obtain mixed nucleic acid;
amplifying the mixed nucleic acid by using the multiple amplification primers to obtain an amplification product, and constructing a high-throughput sequencing library by using the amplification product;
performing high-throughput sequencing on the high-throughput sequencing library to obtain a sequencing fragment group;
analyzing the sequencing fragment group to obtain the number of the sequencing fragments of the drug resistance gene, the number of the sequencing fragments of the endogenous standard gene and the number of the sequencing fragments of the exogenous standard gene in the sample to be detected;
judging whether the experiment is successful or not according to the number of the sequencing fragments of the exogenous standard gene and the number of the sequencing fragments of the endogenous standard gene;
if the experiment is successful, calculating the content of the drug resistance gene in the sample to be tested;
and judging whether the sample to be detected contains the drug resistance gene or not according to the content of the drug resistance gene.
Specifically, the number of drug resistance genes is at least 1, the number of endogenous standard genes is at least 1, and the number of exogenous standard genes is at least 1.
Specifically, the endogenous standard gene is a gene in a microorganism of the sample to be tested.
Specifically, the exogenous standard gene is not present in an organism of known genome.
Specifically, the designed region or the amplified region of the primer for amplifying the drug-resistant gene has homology of less than 98% with the regions other than the drug-resistant gene on the genomes of all the organisms other than the regions of the genomes of the organisms in the test sample.
Specifically, the method for judging whether the experiment is successful or not comprises the steps of successfully performing the experiment when the number of the sequencing fragments of the exogenous standard gene and the number of the sequencing fragments of the endogenous standard gene are both more than or equal to α 1, and failing the experiment when the number of the sequencing fragments of the exogenous standard gene or the number of the sequencing fragments of the endogenous standard gene is less than α 1, wherein α 1 is a judgment threshold.
Specifically, the method for calculating the content of the drug resistance gene in the sample to be detected comprises the following steps: the content of the m-th drug resistance gene is calculated by the formula
Wherein i is the ith test region of the mth drug resistance gene, n1 is the number of test regions of the mth drug resistance gene, bi is the number of sequenced fragments of the ith test region of the mth drug resistance gene, and k is the kth drug resistance geneThe endogenous standard genes, n3 is the number of the endogenous standard genes, j is the jth test region of the kth endogenous standard gene, n2 is the number of the test regions of the kth endogenous standard gene, and aj is the number of sequencing fragments of the jth test region of the kth endogenous standard gene; n is the total number of test regions for all of the endogenous standard genes.
Specifically, the method for judging whether the drug-resistant gene is contained in the sample to be detected comprises the steps of judging that the sample to be detected contains the drug-resistant gene when the content of the drug-resistant gene is not less than α 2, and judging that the sample to be detected does not contain the drug-resistant gene when the content of all the drug-resistant genes is less than α 2, wherein α 2 is a judgment threshold value.
Specifically, the method further comprises: and adding exogenous nucleic acid which cannot be amplified by the multiple amplification primers into the sample to be detected, and extracting the exogenous nucleic acid and the genome of the sample to be detected together.
Specifically, the ratio of the mass of the exogenous standard gene to the total mass of the genome of the test sample is greater than 1/100000.
The technical scheme provided by the embodiment of the invention has the following beneficial effects: the method provided by the embodiment of the invention can be used for detecting any multiple drug resistance genes to be detected in any number and type of microorganisms in a one-time, quantitative, rapid and accurate manner without culture and purification, and can provide comprehensive information required by medication.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below.
Example I detection of drug-resistant Gene of Rice microorganism
The sample to be detected is a rice plant or a part of the rice plant, the sample to be detected in the embodiment is a rice leaf, specifically, the rice leaf used in the embodiment is taken from a paddy field in a Wuhan chaos development area, and the drug resistance gene of the microorganism in the sample to be detected is used for pertinently applying drugs to the rice disease, so that the generation of drug resistance is avoided, meanwhile, unnecessary drugs are reduced, and the cost is saved. The embodiment comprises the following steps:
step 1, determining drug resistance genes of microorganisms needing to be detected in a sample to be detected, endogenous standard genes in the sample to be detected and exogenous standard genes of the sample to be detected, wherein the specific method comprises the following steps:
wherein, the drug resistance genes are at least 1, the endogenous standard genes are at least 1, and the exogenous standard genes are at least 1. The endogenous standard gene is a gene in a microorganism of a sample to be tested. The foreign standard gene is not present in organisms of known genome.
The drug Resistance genes in this example are resistant to penicillin, β -lactamase, cephalosporinase, kanamycin and neomycin, wherein The drug Resistance genes APH (3') -Ia are resistant to kanamycin and neomycin simultaneously, The endogenous standard genes in this example are 1, specifically, The endogenous standard genes are ribosomal rRNA genes which are present in most organisms and have conserved regions, in this example, The exogenous standard genes are 1, specifically, The exogenous standard genes are ERCC-00004 genes, and when they are subjected to homologous alignment at NCBI (http:// www.ncbi.nlm.nih.gov), The presence of homologous sequences in The known organism reference genome is not found, i.e., The absence of The exogenous standard genes in The known organism genome is found, Table 1 is The information and The results of detection of The genes detected in this example, and The names and The sequence numbers of The drug Resistance genes in Table 1 are consistent with The name of The drug Resistance database (CRAD: The Comprehensive Antibiotic Resistance/captasecard).
Table 1 shows the information and the results of the detection of the genes detected in this example
In Table 1, "/" indicates none.
Step 2, preparing a multiplex amplification primer for amplifying test areas of the drug resistance gene, the endogenous standard gene and the exogenous standard gene, wherein the specific method comprises the following steps:
selecting a conserved region of the drug resistance gene to design a multiple amplification primer; the requirement that the designed or amplified region of the primers used to amplify the drug resistance gene have less than 98% homology to other regions of the genome of the organism in the sample to be tested ensures that detection of the drug resistance gene is not interfered with by other regions of the genome of the organism in the sample to be tested. Specifically, the designed region of the primer for the drug-resistant gene is conserved among different variants, so that it is ensured that different variants of the same drug-resistant gene can be amplified using the same primer, and if the homology of the designed region of the primer for the drug-resistant gene with other regions of the genome of the organism in the sample to be tested is less than 98%, the primer does not amplify the other regions, and thus, does not interfere with the detection of the drug-resistant gene. Otherwise, it is required that the amplified region of the primer of the drug-resistant gene has a homology of less than 98% with other regions of the genome of the organism in the sample to be tested, so that the amplified product obtained can be distinguished from the drug-resistant gene without interfering with the detection of the drug-resistant gene as well. Meanwhile, the amplification efficiency of each pair of primers in the multiple amplification primers is between 95% and 105%.
Specifically, the sequences of the drug resistance genes in table 1 were downloaded in an antibiotic resistance database, and the numbering of the sequence of each downloaded drug resistance gene is shown in table 1. And obtaining conserved regions among different sequences of the same drug-resistant gene by utilizing homologous alignment, and using the conserved regions as design regions of the multiple amplification primers. If the drug resistance gene contains only one sequence, the whole region of the sequence is used as a conserved region and a design region of a multiplex amplification primer. And carrying out homologous comparison on the designed region of the multiple amplification primer obtained in the above way and other regions except the drug resistance gene on the genome of all organisms through NCBI, and reserving the designed region of the multiple amplification primer with the homology of less than 98%. The V5 region of the ribosomal rRNA gene and the entire sequence of the ERCC-00004 gene, which is a foreign standard gene, serve as a conserved region and a design region of a multiplex amplification primer.
The process of obtaining the multiple amplification primers is as follows: the log-in Sammer Feishel company multiple PCR primers design webpage https:// ampliseq.com/, and select "DNA Hotspot designs" at the "Application type" option. Connecting the design regions of the obtained multiple amplification primers by using 100N to form an artificial reference genome, and uploading the artificial reference genome after selecting "Custom" from the options of "Select the genome you with to use". The DNA Type option selects "Standard DNA". In the design region of the multiple amplification primers, more than 3 non-overlapping regions are randomly selected as amplification regions and filled in an Add Hotspot option, and finally a 'Submittargets' button is clicked for submission to obtain the multiple amplification primers for amplifying the drug-resistant gene, the endogenous standard gene and the exogenous standard gene. Each primer of the multiple amplification primer sequences and the template sequence amplified by each primer of the multiple amplification primers are synthesized one by Biotechnology engineering (Shanghai) GmbH, wherein the template sequence refers to the amplification region filled with Add Hotspot option, the amplification efficiency of each multiple amplification primer is detected according to the operation manual of StepOne real-time quantitative PCR instrument (Part Number 4376784Rev.E) of Sammer Feishel, USA, and only the multiple amplification primers with the amplification efficiency of 95% -105% are reserved. Multiplex amplification primers were synthesized by seimer feishel, usa and provided in the form of a mixed liquid. This example uses multiplex PCR technology provided by Saimer Feishale, USA, which can amplify up to 12000 test regions simultaneously, therefore, the present invention has the ability to detect all the drug-resistant genes that are hopefully detected at one time.
Step 3, adding exogenous nucleic acid which cannot be amplified by the multiple amplification primers into a sample to be detected, and extracting the exogenous nucleic acid and the genome of the sample to be detected together to obtain a mixed sample, wherein the specific method comprises the following steps:
if the genome content in the sample to be detected is high and the nucleic acid extraction is normal, no exogenous nucleic acid is added. If the content of the genome in the sample to be detected is low and the extraction of the genome is difficult, the genome in the sample to be detected can be conveniently extracted after the exogenous nucleic acid which cannot be amplified by the multiple amplification primers is added into the sample to be detected, and the normal extraction of the genome in the sample to be detected can be ensured by adding about 1ug of the exogenous nucleic acid under the general condition. The amount of the exogenous nucleic acid to be added may be appropriately adjusted depending on the method of nucleic acid extraction, and for example, in the case of large-scale extraction, the amount of the exogenous nucleic acid to be added may be increased, and in the case of ultra-fine extraction, the amount of the exogenous nucleic acid to be added may be appropriately decreased, and in the case of the existing genome extraction technology, the amount of the exogenous nucleic acid to be added is generally not less than 1ng at minimum. Since the multiple amplification primers cannot amplify the foreign nucleic acid, the added foreign nucleic acid does not affect the detection of the drug-resistant gene. In this example, the exogenous nucleic acid is ERCC-00024 gene, the sequence of the exogenous nucleic acid ERCC-00024 gene is shown by SEQ ID NO:13 in the sequence Listing, and the ERCC-00024 gene is synthesized by Biotechnology engineering (Shanghai) GmbH. In this example, when the genome was extracted using the N96 plant genomic DNA extraction kit, it was found that the amount of nucleic acid extracted from the sample to be tested was normal, and therefore, no exogenous nucleic acid was added to the sample to be tested.
And 4, extracting the genome of the sample to be detected, wherein the specific method comprises the following steps:
the genome of the sample to be tested is extracted according to the operation instruction of an N96 rice genome DNA extraction kit (production company: Tiangen Biochemical technology (Beijing) Co., Ltd., product goods number: DP338), and the extracted genome nucleic acid is the genome nucleic acid of the sample to be tested. The amount of the genome of the obtained sample to be tested was measured by using a double-stranded DNA program in a spectrophotometer (model Q5000, manufactured by Quawell, USA). In this example, the total amount of the genome of the test sample was 3442 ng.
Step 5, adding an exogenous standard gene into the genome of the sample to be detected, wherein the ratio of the mass of the exogenous standard gene to the total mass of the genome of the sample to be detected is greater than 1/100000, and obtaining the mixed nucleic acid, wherein the specific method comprises the following steps:
in this example, the ratio of the mass of the exogenous standard gene to the total mass of the genome of the test sample was 1/1000, and since the total amount of the genome of the test sample was 3442ng, 3.442ng of the exogenous standard gene was added to obtain a mixed nucleic acid.
Step 6, amplifying the mixed nucleic acid by using the multiple amplification primers to obtain an amplification product, and constructing a high-throughput sequencing library by using the amplification product, wherein the specific method comprises the following steps:
the library construction kit 2.0 (manufactured by seimer feishel, usa, cat # 4475345) was used to construct a high-throughput sequencing library. The library construction kit comprises the following reagents: 5 × Ion AmpliSeqTMHiFi Mix, FuPa reagent, conversion reagent, sequencing linker solution, and DNA ligase. Method for constructing library according to operation manual of library construction kit IonAmpliSeqTMLibrary Preparation (publication number: MAN0006735, version: A.0). The multiplex PCR amplification system is as follows: 5 × Ion AmpliSeqTMHiFi Mix 4 ul, mixed liquid of prepared multiplex amplification primers 4 ul, mixed nucleic acid 10ng and enzyme-free water 11 ul. The amplification procedure for multiplex PCR was as follows: 99 ℃ for 2 minutes; (99 ℃, 15 seconds; 60 ℃, 4 minutes) x 25 cycles; keeping the temperature at 10 ℃. After redundant primers in the multiple PCR amplification product are digested by a FuPa reagent, phosphorylation is carried out, and the specific method comprises the following steps: adding 2 ul FuPa reagent into the amplification product of the multiplex PCR, mixing evenly, and reacting on a PCR instrument according to the following program: 10 minutes at 50 ℃; at 55 ℃ for 10 minutes; 10 minutes at 60 ℃; storing at 10 ℃ to obtain a mixture a, wherein the mixture a is a solution containing the phosphorylated amplification product. Connecting the phosphorylated amplification product with a sequencing adaptor by the following specific method: mu.l of the conversion reagent, 2. mu.l of the sequencing adapter solution and 2. mu.l of the DNA ligase were added to the mixture a, mixed and reacted on a PCR instrument according to the following procedure: 30 minutes at 22 ℃; 72 ℃ for 10 minutes; storing at 10 ℃ to obtain a mixed solution b. The mixture b was purified by standard ethanol precipitation and dissolved in 10. mu.l of enzyme-free water. Manufactured by Invitrigen corporation of America
detecting the dsDNA HS Assay Kit (cat No. Q32852) according to the instruction to obtain the mass concentration of the mixed solution b, and purifying the purified mixed solution bMix b was diluted to 15ng/ml to give a high throughput sequencing library at a concentration of about 100 pM.
And 7, performing high-throughput sequencing on the high-throughput sequencing library to obtain a sequencing fragment group, wherein the specific method comprises the following steps:
the obtained high-throughput sequencing library and a Kit Ion PI Template OT 2200 Kit v2 (manufactured by Invirrrigen, USA, with the product number of 4485146) are used for ePCR (Emulsion PCR) amplification before sequencing, and the operation method is carried out according to the operation manual of the Kit. High-throughput sequencing was performed on a Proton second generation high-throughput sequencer using the ePCR product and a Kit Ion PIsequencing 200Kit v2 (manufactured by Invirriggen, USA, Cat. No. 4485149), and the procedure was performed according to the manual of the Kit. In this example, the high throughput sequencing amount is set as 1M sequencing fragment (1M ═ 100 ten thousand), the sequencing length is set as 500 cycles, and after the sequencing is finished, the sequencing fragment group is obtained.
And 8, analyzing the sequencing fragment group to obtain the number of the sequencing fragments of the drug resistance gene, the number of the sequencing fragments of the endogenous standard gene and the number of the sequencing fragments of the exogenous standard gene in the sample to be detected, wherein the specific method comprises the following steps:
according to the primers of the sequencing fragments, blastall (version 2.2.26) software is utilized, according to default parameter settings, the sequencing fragment groups are compared to the detection areas of the drug resistance gene, the endogenous standard gene and the exogenous standard gene corresponding to the multiple amplification primers in the table 1, the sequencing fragments with homology higher than 98% with the detection areas (the sequencing fragments with homology lower than 98% may be obtained by non-specific amplification) are reserved, and respectively represent the number of the sequencing fragments of the drug resistance gene, the number of the sequencing fragments of the endogenous standard gene and the number of the sequencing fragments of the exogenous standard gene in a sample to be detected, and the results are shown in the table 1.
Step 9, judging whether the experiment is successful according to the number of the sequencing fragments of the exogenous standard genes and the number of the sequencing fragments of the endogenous standard genes, wherein the specific method comprises the following steps:
the method for judging whether the experiment is successful or not comprises the steps of judging whether the experiment is successful or not when the number of sequencing fragments of the exogenous standard genes and the number of sequencing fragments of the endogenous standard genes are both more than or equal to α 1, judging whether the experiment is successful or not when the number of sequencing fragments of the exogenous standard genes or the number of sequencing fragments of the endogenous standard genes is less than α 1, judging whether the experiment is failed or not, wherein α 1 is a judgment threshold value, if the number of the exogenous standard genes is too low, high-throughput library construction or high-throughput sequencing is possible, if the number of the endogenous standard genes is too low, nucleic acid extraction is possible, and if the experiment is unsuccessful, adjusting specific conditions until the experiment is successful, in the embodiment, α 1 takes 10 sequencing fragments, and as can be seen from table 1, the number of the sequencing fragments of the exogenous standard genes and the number of the sequencing fragments of the endogenous standard genes are both more than or not more than 10, so that the experiment provided by the embodiment.
Step 10, if the experiment is successful, calculating the content of the drug resistance gene in the sample to be tested, wherein the specific method comprises the following steps:
the content of the m drug resistance gene is calculated by the formula
Wherein i is the ith test region of the mth drug resistance gene, n1 is the number of the test regions of the mth drug resistance gene, bi is the number of sequencing fragments of the ith test region of the mth drug resistance gene, k is the kth endogenous standard gene, n3 is the number of endogenous standard genes, j is the jth test region of the kth endogenous standard gene, n2 is the number of the test regions of the kth endogenous standard gene, and aj is the number of sequencing fragments of the jth test region of the kth endogenous standard gene; n is the total number of test regions for all endogenous standard genes. The method adopts the average number to calculate the content of the drug-resistant gene, aims to reduce the influence of factors such as amplification efficiency on the result, and has little influence on the result if the amplification efficiency of each multiple amplification primer is detected and the amplification efficiency is ensured to be between 95 percent and 105 percent, so that the drug-resistant gene, the endogenous standard gene and the exogenous standard gene can be represented by only one detection region amplified by the multiple amplification primers.
As can be seen from table 1, in this example, 4 drug resistance genes were detected in total, so that m is 4, and each drug resistance gene detected 1 test region, so that N1 is 1, and 1 endogenous standard gene was detected in total, so that N3 is 1, and each endogenous standard gene detected 1 test region in total, so that N2 is 1, and N is 1, and table 1 lists the number of sequencing fragments of each drug resistance gene and each endogenous standard gene, and substituting them into the calculation formula of the content of drug resistance genes can obtain the content of each drug resistance gene, and the results are shown in table 1.
Step 11, judging whether the sample to be detected contains the drug resistance gene according to the content of the drug resistance gene, wherein the specific method comprises the following steps:
the method for determining that the sample to be tested contains the drug resistance genes comprises the steps of determining that the sample to be tested contains the drug resistance genes when the content of the drug resistance genes is larger than or equal to α 2, determining that the sample to be tested does not contain the drug resistance genes when the content of all the drug resistance genes is smaller than α 2, wherein α 2 is a determination threshold value α 2 is set artificially according to the required severity, when a small amount of drug resistance genes exist and antibiotics are ineffective, α 2 is lower, but higher, when the amount of the drug resistance genes lack is related to the effects of the antibiotics (most of the cases are the same), α 2 is set to 0.1% so as to balance statistical errors and sensitivity, in the embodiment, α 2 is set to 0.1%, as can be seen from table 1, in the embodiment, when the content of three drug resistance genes except for the drug resistance genes CTX-M-14 is smaller than α 2, which is 0.1%, therefore, the sample to be determined as containing the drug resistance genes because the corresponding drug resistance genes CTX-M-14 exist, the content of the drug resistance genes of the rice can be quantified and the drug resistance genes can be detected by a quantitative amplification primer 4, and the quantitative detection of multiple drug resistance genes can be realized only once.
The detection result of this embodiment is verified, and the specific method is as follows:
the multiple amplification primers in table 1 were synthesized manually, each multiple amplification primer was used to perform PCR amplification on the DNA of the sample to be tested one by one, the amplification product was subjected to agarose electrophoresis detection, it was found that there was an amplification band in the drug-resistant gene CTX-M-14, and it was judged as positive, meanwhile, there were significant amplification bands in both the endogenous standard gene and the exogenous standard gene, and it was judged as positive, and the amplification products of the primers for the other three drug-resistant genes had no significant band, and it was judged as negative, which was consistent with the detection results of this example.
Example II detection of drug-resistant genes in corn microorganisms
The corn leaves used in this example are taken from a field of corn in a chaos opening development area in wuhan, and the drug resistance genes of the microorganisms in the field are detected in order to specifically administer drugs to the corn diseases, thereby preventing the generation of drug resistance, and reducing unnecessary drugs and saving cost. The embodiment comprises the following steps:
determining the drug resistance gene of the microorganism to be detected in the sample to be detected, the endogenous standard gene in the sample to be detected and the exogenous standard gene of the sample to be detected according to the same method as the first embodiment; preparing a multiplex amplification primer for amplifying test regions of the drug resistance gene, the endogenous standard gene and the exogenous standard gene; the results are shown in Table 2.
Table 2 shows the information and the results of the detection of the genes detected in this example
Adding exogenous nucleic acid which cannot be amplified by the multiple amplification primers into a sample to be detected according to a method similar to that of the first embodiment, and extracting the exogenous nucleic acid and the genome of the sample to be detected together to obtain a mixed sample; extracting the genome of the mixed sample, wherein the total amount of the genome of the obtained mixed sample is 3520 ng; the exogenous standard gene is added into the genome of the mixed sample, the ratio of the mass of the exogenous standard gene to the total mass of the genome of the mixed sample is 1/1000, and the total amount of the genome of the mixed sample is 3520ng, so 3.52ng of the exogenous standard gene needs to be added to obtain the mixed nucleic acid.
Amplifying the mixed nucleic acid by using multiple amplification primers according to the same method as the first embodiment to obtain an amplification product, and constructing a high-throughput sequencing library by using the amplification product; performing high-throughput sequencing on the high-throughput sequencing library to obtain a sequencing fragment group; judging that the experiment provided by the embodiment is successful according to the number of the sequencing fragments of the exogenous standard gene and the number of the sequencing fragments of the endogenous standard gene; the content of the drug-resistant gene in the sample to be tested was calculated and the results are shown in table 2.
In this example, the content of all three drug-resistant genes except the drug-resistant gene CTX-M-14 in this example was judged to be < α 2 ═ 0.1% by the same method as in example one, and therefore, it was judged that the sample to be tested contained the drug-resistant gene CTX-M-14.
The method provided by the embodiment of the invention can be used for detecting different samples to be detected, has strong universality, and is mainly different in that a corresponding genome extraction method is selected for extracting the genome according to different samples to be detected. Sequencing is the ultimate standard of nucleic acid detection, so the detection result of the embodiment of the invention has high accuracy; sequencing can distinguish single base difference, so that the drug resistance gene detection in the embodiment of the invention has high resolution. The method provided by the embodiment of the invention can be used for detecting on the premise of no need of culture and purification, so that the detection is quick, which is very important for timely diagnosis and timely medication of the disease condition, and simultaneously, the possibility is provided for detecting the non-culturable pathogens. The embodiment of the invention can detect any multiple drug resistance genes and provides comprehensive information for searching an antibiotic for effectively preventing or treating plant diseases. Therefore, the embodiment of the invention can detect any plurality of drug resistance genes which are expected to be detected in any number and types of microorganisms in a one-time, quantitative, rapid and accurate manner, and the effect is not achieved by the prior art.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (5)
1. A method for detecting a microbial drug resistance gene in a plant, comprising:
determining a drug resistance gene of a microorganism to be detected in a sample to be detected, an endogenous standard gene in the sample to be detected and an exogenous standard gene of the sample to be detected, wherein the sample to be detected is a whole plant or a part of the plant;
preparing multiplex amplification primers for amplifying test regions of the drug resistance gene, the endogenous standard gene and the exogenous standard gene;
adding exogenous nucleic acid incapable of being amplified by the multiple amplification primers into the sample to be detected, extracting the genome of the sample to be detected, and extracting the exogenous nucleic acid and the genome of the sample to be detected together;
adding the exogenous standard gene into the genome of the sample to be detected to obtain mixed nucleic acid;
amplifying the mixed nucleic acid by using the multiple amplification primers to obtain an amplification product, and constructing a high-throughput sequencing library by using the amplification product;
performing high-throughput sequencing on the high-throughput sequencing library to obtain a sequencing fragment group;
analyzing the sequencing fragment group to obtain the number of the sequencing fragments of the drug resistance gene, the number of the sequencing fragments of the endogenous standard gene and the number of the sequencing fragments of the exogenous standard gene in the sample to be detected, wherein the ratio of the mass of the exogenous standard gene to the total mass of the genome of the sample to be detected is greater than 1/100000;
judging whether the experiment succeeds or not according to the number of the sequencing fragments of the exogenous standard gene and the number of the sequencing fragments of the endogenous standard gene, wherein the method for judging whether the experiment succeeds or not is that when the number of the sequencing fragments of the exogenous standard gene and the number of the sequencing fragments of the endogenous standard gene are both more than or equal to α 1, the experiment succeeds, when the number of the sequencing fragments of the exogenous standard gene or the number of the sequencing fragments of the endogenous standard gene is less than α 1, the experiment fails, wherein α 1 is a judgment threshold value;
if the experiment is successful, calculating the content of the drug-resistant gene in the sample to be tested, wherein the method for calculating the content of the drug-resistant gene in the sample to be tested comprises the following steps: the content of the m-th drug resistance gene is calculated by the formula
Wherein i is the ith test region of the mth drug resistance gene, n1 is the number of the test regions of the mth drug resistance gene, bi is the number of the sequencing fragments of the ith test region of the mth drug resistance gene, k is the kth endogenous standard gene, n3 is the number of the endogenous standard genes, j is the jth test region of the kth endogenous standard gene, n2 is the number of the test regions of the kth endogenous standard gene, and aj is the number of the sequencing fragments of the jth test region of the kth endogenous standard gene; n is the total number of test regions of all the endogenous standard genes;
judging whether the sample to be detected contains a drug-resistant gene or not according to the content of the drug-resistant gene, wherein the method for judging whether the sample to be detected contains the drug-resistant gene is to judge that the sample to be detected contains the drug-resistant gene when the content of the drug-resistant gene is more than or equal to α 2, and judge that the sample to be detected does not contain the drug-resistant gene when the content of all the drug-resistant genes is less than α 2, wherein α 2 is a judgment threshold value.
2. The method of claim 1, wherein the number of drug resistance genes is at least 1, the number of endogenous standard genes is at least 1, and the number of exogenous standard genes is at least 1.
3. The method according to claim 1, wherein the endogenous standard gene is a gene in a microorganism of the sample to be tested.
4. The detection method according to claim 1, wherein the exogenous standard gene is not present in an organism having a known genome.
5. The method of claim 1, wherein the designed or amplified region of the primer for amplifying the drug resistance gene has less than 98% homology with other regions of the genome of the organism in the test sample.
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| CN102181575A (en) * | 2011-03-18 | 2011-09-14 | 中国科学院武汉病毒研究所 | Primers and method for detecting drug resistance mutation site of hepatitis B virus |
| CN104561350A (en) * | 2015-01-28 | 2015-04-29 | 深圳华大基因研究院 | Kit and application thereof |
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| CN102181575A (en) * | 2011-03-18 | 2011-09-14 | 中国科学院武汉病毒研究所 | Primers and method for detecting drug resistance mutation site of hepatitis B virus |
| CN104561350A (en) * | 2015-01-28 | 2015-04-29 | 深圳华大基因研究院 | Kit and application thereof |
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