CN105624297A - Method for detecting drug-resistance genes of microorganisms in plants - Google Patents
Method for detecting drug-resistance genes of microorganisms in plants Download PDFInfo
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Abstract
The invention discloses a method for detecting the drug-resistance genes of microorganisms in plants. The method includes: determining the drug-resistance genes, endogenous standard genes and exogenous standard genes; preparing multiple amplification primers; adding exogenous nucleic acid into a to-be-detected sample to obtain a mixed sample, and extracting genome; adding the exogenous standard genes into the genome to obtain mixed nucleic acid; amplifying the mixed nucleic acid, using the amplification product to build a high-throughput sequencing library, and performing high-throughput sequencing to obtain a sequencing fragment group; analyzing the sequencing fragment group, and judging whether the experiment is successful or not according to the number of the acquired sequencing fragments of the exogenous standard genes and the number of the acquired sequencing fragments of the endogenous standard genes; if so, calculating the content of the drug-resistance genes; judging whether the to-be-detected sample contains the drug-resistance genes or not according to the content of the drug-resistance genes. The method has the advantages that optional multiple drug-resistance genes in optional microorganisms can be quantitatively detected in one step, the detection does not need microorganism culture or purification, and the method is fast and accurate and reliable in result.
Description
Technical field
The present invention relates to biological technical field, particularly to the detection method of a kind of phytomicroorganism drug resistance gene.
Background technology
Antibiotics is widely used in controlling plant diseases, current plant disease is in the process of preventing and treating, and abuse of antibiotics class Phenomenon of Drugs is very general, causes pathogenic microorganism to be evolved out drug resistance gene, produce drug resistance so that antibiotics lost efficacy when controlling plant diseases. Accordingly, it would be desirable to according to microorganism drug resistance gene medication pointedly in plant, to avoid pathogen to produce drug resistance, meanwhile, reduce pesticide dosage, reduce cost.
The method of existing detection drug resistance gene includes: estimate the pathogen of the drug resistance gene of the needs detection carried on testing sample, separate and this pathogen of purification, by PCR (PolymeaseChainReaction, polymerase chain reaction) this drug resistance gene of primer amplification, utilize electrophoresis or real time quantitative PCR method judge whether there is the drug resistance gene needing detection in testing sample one by one.
In the process realizing the present invention, inventor have found that prior art at least exists the one in problems with:
Need to cultivate, separate and purification pathogen so that the time of detection is longer, delay treatment opportunity; The drug resistance gene in not educable fastbacteria cannot be detected; Once can only detect for a kind of or a few pathogen; Once can only detect one or a few drug resistance gene, it is impossible to all of drug resistance gene in testing sample detected, it is impossible to give the comprehensive information needed for medication; The detection by quantitative of drug resistance gene can not be realized.
Summary of the invention
In order to solve that prior art detects the problem that the method for drug resistance gene has much room for improvement, embodiments provide the detection method of a kind of phytomicroorganism drug resistance gene. Described technical scheme is as follows:
Embodiments providing the detection method of a kind of phytomicroorganism drug resistance gene, described method includes:
Determining the external source standard gene of the endogenous standard gene and described testing sample needed in testing sample in the drug resistance gene of microorganism of detection, described testing sample, described testing sample is whole plant plant or the described plant of part;
Preparation is for expanding the multiplex amplification primer of the test zone of described drug resistance gene, described endogenous standard gene and described external source standard gene;
Extract the genome of described testing sample;
Described external source standard gene is added, it is thus achieved that mixing nucleic acid in the genome of described testing sample;
Utilize mixing nucleic acid described in described multiplex amplification primer pair to expand, obtain amplified production, utilize described amplified production to build high-throughput sequencing library;
Described high-throughput sequencing library is carried out high-flux sequence, obtains order-checking fragment group;
Analyze described order-checking fragment group, it is thus achieved that the quantity of the order-checking fragment of the quantity of the order-checking fragment of drug resistance gene described in described testing sample, the quantity of order-checking fragment of described endogenous standard gene and described external source standard gene;
The quantity of the quantity of the order-checking fragment according to described external source standard gene and the order-checking fragment of described endogenous standard gene, it is judged that whether experiment is successful;
If described Success in Experiment, then calculate the content of drug resistance gene described in described testing sample;
Content according to described drug resistance gene judges whether described testing sample contains drug resistance gene.
Specifically, described drug resistance gene is at least 1, and described endogenous standard gene is at least 1, and described external source standard gene is at least 1.
Specifically, described endogenous standard gene is the gene in the microorganism of described testing sample.
Specifically, described external source standard gene is not present in the biology of known group.
Specifically, for expand biological other region genomic in the design section of the primer of described drug resistance gene or amplification region and described testing sample on zoic genome the homology in other region except drug resistance gene lower than 98%.
Specifically, described judgment experiment whether successfully method is: when the quantity of the quantity of order-checking fragment of described external source standard gene and the order-checking fragment of described endogenous standard gene equal>=�� 1 time, then Success in Experiment; When the quantity of order-checking fragment of described external source standard gene or the order-checking fragment of described endogenous standard gene quantity<during �� 1, then the failure of an experiment; Wherein, �� 1 is decision threshold.
Specifically, the method for the content of drug resistance gene described in described testing sample that calculates is: described in m kind, the computing formula of the content of drug resistance gene isWherein, i is the i-th test zone of drug resistance gene described in m kind, n1 is the number of the test zone of drug resistance gene described in described m kind, bi is the quantity of the order-checking fragment of the i-th test zone of described m kind drug resistance gene, k is endogenous standard gene described in kth kind, n3 is the number of described endogenous standard gene, j is the jth test zone of endogenous standard gene described in kth kind, n2 is the number of the test zone of endogenous standard gene described in kth kind, and aj is the quantity of the order-checking fragment of the jth kind test zone of endogenous standard gene described in kth kind; N is the sum of the test zone of all described endogenous standard genes.
Specifically, it is determined that the method whether containing described drug resistance gene in described testing sample is: during the content>=�� 2 of described drug resistance gene, it is determined that described testing sample contains described drug resistance gene; When the content of all described drug resistance genes is<during �� 2, it is determined that described testing sample is without there being described drug resistance gene; Wherein, �� 2 is decision threshold.
Specifically, described method also includes: adds the exogenous nucleic acid that described multiplex amplification primer can not expand in described testing sample, is together extracted by the genome of described exogenous nucleic acid Yu described testing sample.
Specifically, the quality of described external source standard gene and the ratio of the genomic gross mass of described testing sample are more than 1/100000.
The technical scheme that the embodiment of the present invention provides has the benefit that the method that the embodiment of the present invention provides can under need not cultivating the premise with purification, any multiple drug resistance gene wishing detection disposable, quantitative, quickly, in the microorganism of detection any amount and type accurately, it is possible to give the comprehensive information needed for medication.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearly, below embodiment of the present invention is described further in detail.
Embodiment one, Oryza sativa L. microorganism drug resistance gene detection
Testing sample is a part for rice plant or rice plant, in the present embodiment, testing sample is rice leaf, specifically, the rice leaf used in the present embodiment takes from the rice terrace in Zhuankou development zone, Wuhan, the drug resistance gene detecting microorganism therein is in order to pointedly to rice disease medication, it is to avoid the generation of drug resistance, simultaneously, reduce unnecessary medication, save cost. The present embodiment comprises the steps:
Step 1, determining the external source standard gene needing the drug resistance gene of microorganism of detection, the endogenous standard gene in testing sample and testing sample in testing sample, concrete grammar is as follows:
Wherein, drug resistance gene is at least 1, and endogenous standard gene is at least 1, and external source standard gene is at least 1. Endogenous standard gene is the gene in the microorganism of testing sample. External source standard gene is not present in the biology of known group.
Drug resistance gene resistance to penicillin in the present embodiment, beta-lactamase, cephalosporinase, kanamycin and neomycin, wherein, drug resistance gene APH (3')-Ia anti-kanamycin and neomycin simultaneously. In the present embodiment, endogenous standard gene is a kind, and specifically, endogenous standard gene is ribosome rRNA gene, and this ribosome rRNA gene is present in overwhelming majority biology and has conservative region. In the present embodiment, external source standard gene is a kind, specifically, external source standard gene is ERCC-00004 gene, when it carries out homology comparison on NCBI (http://www.ncbi.nlm.nih.gov), not finding that it exists homologous sequence in known reference biomolecule genome, namely external source standard gene is not present in known biological genome. Table 1 is detected gene-correlation information and testing result in the present embodiment, drug resistance gene title and sequence numbering are consistent with sequence numbering with the title in antibiotic resistance data base (CRAD:TheComprehensiveAntibioticResistanceDatabase, network address is http://arpcard.mcmaster.ca/) in Table 1.
Table 1 is detected gene-correlation information and testing result in the present embodiment
In table 1 "/" indicate without.
Step 2, preparation are for expanding the multiplex amplification primer of the test zone of drug resistance gene, endogenous standard gene and external source standard gene, and concrete grammar is as follows:
Select the conservative region design multiplex amplification primer of drug resistance gene; For expanding the homology in biological other region genomic in the design section of the primer of drug resistance gene or amplification region and testing sample lower than 98%, this requirement ensure that the detection of drug resistance gene is by the interference in biological other region genomic in testing sample. Specifically, the design section of the primer of drug resistance gene is conservative between different variants, such guarantee can amplify the different variants of identical drug resistance gene with identical primer, if the homology in biological other region genomic in the design section of the primer of drug resistance gene and testing sample is lower than 98%, then this primer will not expand other region, therefore, without interference with the detection of drug resistance gene. Otherwise, the homology in biological other region genomic in the amplification region of the primer of requirement drug resistance gene and testing sample is lower than 98%, so, it is thus achieved that amplified production can distinguish mutually with drug resistance gene, equally without interference with the detection of drug resistance gene. Meanwhile, in multiplex amplification primer the amplification efficiency of every pair of primer all between 95%��105%.
Specifically, the sequence of the drug resistance gene in download table 1 in antibiotic resistance data base, the numbering of the sequence of the drug resistance gene of each download is in Table 1. Homology comparison is utilized to obtain the conservative region between the different sequences of same drug resistance gene, as the design section of multiplex amplification primer. If drug resistance gene only comprises a kind of sequence, then the Zone Full of this sequence is as the design section of conservative region and multiplex amplification primer. The design section of multiplex amplification primer achieved above is carried out homology comparison, the design section of the reservation homology multiplex amplification primer lower than 98% by NCBI with other region except drug resistance gene on institute zoic genome. The V5 district of ribosome rRNA gene and the full sequence of external source standard gene ERCC-00004 gene are as the design section of conservative region and multiplex amplification primer.
Multiplex amplification primer acquisition process is as follows: logs in match Mo Feishier company multiple PCR primer Photographing On-line webpage https: //ampliseq.com/, selects " DNAHotspotdesigns (single-pool) " at " Applicationtype " option. the design section of the multiplex amplification primer of acquisition is coupled together with 100 N, forms an artificial reference genome, and in " Selectthegenomeyouwishtouse " option after selection " Custom ", upload artificial reference genome. DNAType option selects " StandardDNA ". in the design section of multiplex amplification primer, nonoverlapping region of random choose more than 3 is as amplification region, and insert in AddHotspot option, finally click " Submittargets " button to submit to, it is thus achieved that for expanding the multiplex amplification primer of drug resistance gene, endogenous standard gene and external source standard gene. the each heavy primer of multiplex amplification primer sequence is synthesized one by one by Sangon Biotech (Shanghai) Co., Ltd., and the template sequence expanded by each heavy primer of multiplex amplification primer, template sequence refers to that above-mentioned multiplex amplification primer is in the amplification region inserting AddHotspot option, the workbook (PartNumber4376784Rev.E) matching the StepOne real-time PCR of Mo Feishier company according to the U.S. detects the amplification efficiency of each multiplex amplification primer, only retain the multiplex amplification primer that amplification efficiency is 95%��105%, in the present embodiment, for saving cost, in the multiplex amplification primer satisfied condition, only choose closest to 100% a primer as final selected multiplex amplification primer, result is in Table 1. multiplex amplification primer is matched the synthesis of Mo Feishier company by the U.S. and the form to mix liquid provides and uses. the present embodiment adopt the U.S. match Mo Feishier company provide multiple PCR technique, it can expand up to 12000 test zones simultaneously, therefore, the present invention have the ability disposable detection existing be hopeful detection drug resistance gene.
Step 3, the exogenous nucleic acid that addition multiplex amplification primer can not expand in testing sample, together extract the genome of exogenous nucleic acid Yu testing sample, obtain biased sample, and concrete grammar is as follows:
If genome content is higher in testing sample, nucleic acid extraction is normal, then need not add exogenous nucleic acid. If genomic content is relatively low in testing sample, genome extracts difficulty, after then needing the exogenous nucleic acid that addition multiplex amplification primer can not expand in testing sample, can conveniently extract the genome in testing sample, generally, need to add the exogenous nucleic acid of about 1ug, the genomic normal extraction in testing sample can be ensured. The amount of the exogenous nucleic acid added can appropriately adjust according to the extracting method of nucleic acid, for instance, when extracting in a large number, addition should increase to some extent, when ultramicron is extracted, it is possible to suitably reduce, under existing genome extractive technique level, generally minimum must not less than 1ng. Owing to multiplex amplification primer can not expand exogenous nucleic acid, therefore, affiliated exogenous nucleic acid has no effect on the detection of drug resistance gene. In the present embodiment, exogenous nucleic acid is ERCC-00024 gene, and the sequence of exogenous nucleic acid ERCC-00024 gene is such as shown in SEQ ID NO:13, and ERCC-00024 gene is synthesized by Sangon Biotech (Shanghai) Co., Ltd.. In the present embodiment, when adopting N96 plant genome DNA to extract test kit extraction genome, it has been found that the nucleic acid amount extracted from testing sample is normal, therefore, the exogenous nucleic acid that need not add in testing sample.
Step 4, extract testing sample genome, concrete grammar is as follows:
Extract test kit by N96 oryza sativa genomic dna and (produce company: TIANGEN Biotech (Beijing) Co., Ltd., product article No.: DP338) operating instruction extract the genome of testing sample, extract for the genomic nucleic acids of testing sample. Utilize the double-stranded DNA program in spectrophotometer (Quawell company of the U.S. produces, and model is Q5000), the genomic amount of the testing sample that detection obtains. In the present embodiment, the genomic total amount of testing sample is 3442ng.
Step 5, adding external source standard gene in the genome of testing sample, the ratio of the genomic gross mass of the quality of external source standard gene and testing sample is more than 1/100000, it is thus achieved that mixing nucleic acid, and concrete grammar is as follows:
In the present embodiment, the ratio of the genomic gross mass of the quality of external source standard gene and testing sample is 1/1000, owing to the genomic total amount of testing sample is 3442ng, accordingly, it would be desirable to add 3.442ng external source standard gene, it is thus achieved that mixing nucleic acid.
Step 6, utilizing multiplex amplification primer pair mixing nucleic acid to expand, obtain amplified production, utilize amplified production to build high-throughput sequencing library, concrete grammar is as follows:
Library construction Kit 2.0 (being matched Mo Feishier company by the U.S. to produce, article No. is 4475345) is utilized to build high-throughput sequencing library. This library construction Kit includes following reagent: 5 �� IonAmpliSeqTMHiFiMix, FuPa reagent, transferring reagent, sequence measuring joints solution and DNA ligase. The method of library construction is by the workbook " IonAmpliSeq of this library construction KitTMLibraryPreparation " (publication number: MAN0006735, version: A.0) carry out. The amplification system of multiplex PCR is as follows: 5 �� IonAmpliSeqTMHiFiMix4 �� l, the mixing liquid 4 �� l of multiplex amplification primer of preparation, mixing nucleic acid 10ng and without enzyme water 11 �� l. The amplification program of multiplex PCR is as follows: 99 DEG C, 2 minutes; (99 DEG C, 15 seconds; 60 DEG C, 4 minutes) �� 25 circulations; 10 DEG C of insulations. After utilizing FuPa reagent to digest primer unnecessary in multiplexed PCR amplification product, then carry out phosphorylation, method particularly includes: in the amplified production of multiplex PCR, add 2 �� lFuPa reagent, after mixing, by the reaction of following program in PCR instrument: 50 DEG C, 10 minutes; 55 DEG C, 10 minutes; 60 DEG C, 10 minutes; 10 DEG C of preservations, obtaining mixture a, mixture a is containing the amplified production solution through phosphorylation. The amplified production of phosphorylation is connected upper sequence measuring joints, method particularly includes: in mixture a, add transferring reagent 4 �� l, sequence measuring joints solution 2 �� l and DNA ligase 2 �� l, after mixing, by the reaction of following program in PCR instrument: 22 DEG C, 30 minutes; 72 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixed liquor b. It is dissolved in 10 �� l without in enzyme water after utilizing the ethanol precipitation methods purification mixed liquor b of standard. American I nvitrigen company is utilized to produceDsDNAHSAssayKit (article No. is Q32852) also detects according to its description, it is thus achieved that after the mass concentration of mixed liquor b, the mixed liquor b after purification is diluted to 15ng/ml, obtains concentration and be about the high-throughput sequencing library of 100pM.
Step 7, high-throughput sequencing library being carried out high-flux sequence, obtain order-checking fragment group, concrete grammar is as follows:
(invirtrigen company of the U.S. produces to utilize the high-throughput sequencing library obtained and test kit IonPITemplateOT2200Kitv2, article No. is 4485146) check order before ePCR (EmulsionPCR, emulsion polymerization enzyme chain reaction) amplification, operational approach is undertaken by the workbook of this test kit. (invirtrigen company of the U.S. produces to utilize ePCR product and test kit IonPISequencing200Kitv2, article No. is 4485149) on the secondary high-flux sequence instrument of Proton, carry out high-flux sequence, operational approach is undertaken by the workbook of this test kit. In the present embodiment, high-flux sequence amount is set to 1M order-checking fragment (1M=,100 ten thousand), and order-checking length is set to 500cycle (circulation), after order-checking terminates, it is thus achieved that order-checking fragment group.
Step 8, analyzing order-checking fragment group, it is thus achieved that the quantity of the order-checking fragment of the quantity of the order-checking fragment of the quantity of the order-checking fragment of drug resistance gene, endogenous standard gene and external source standard gene in testing sample, concrete grammar is as follows:
Primer according to order-checking fragment, utilize blastall (version2.2.26) software, arrange by the parameter of its acquiescence, the drug resistance gene that multiplex amplification primer pair in order-checking fragment group comparison to table 1 is answered, on the detection region of endogenous standard gene and external source standard gene, retain the order-checking fragment (being likely to be obtained with coming from the order-checking fragment lower than 98%) higher than 98% of the homology with detection region by non-specific amplification, they represent the quantity of the order-checking fragment of drug resistance gene in testing sample respectively, the quantity of the quantity of the order-checking fragment of endogenous standard gene and the order-checking fragment of external source standard gene, its result is in Table 1.
Step 9, quantity according to the quantity of the order-checking fragment of external source standard gene and the order-checking fragment of endogenous standard gene, it is judged that experiment whether success, concrete grammar is as follows:
Judgment experiment whether successfully method is: when the quantity of the quantity of order-checking fragment of external source standard gene and the order-checking fragment of endogenous standard gene equal>=�� 1, then Success in Experiment; When the quantity of order-checking fragment of external source standard gene or the order-checking fragment of endogenous standard gene quantity<during �� 1, then the failure of an experiment; Wherein, �� 1 is decision threshold. If the amount of external source standard gene is too low, it may be possible to high flux library construction or high-flux sequence are unsuccessful, if endogenous standard gene amount is too low, it may be possible to nucleic acid extraction is unsuccessful, if testing unsuccessful, is adjusted in light of the circumstances, till Success in Experiment. In the present embodiment, �� 1 takes 10 order-checking fragments, as it can be seen from table 1 the quantity of the order-checking fragment of the quantity of the order-checking fragment of external source standard gene and endogenous standard gene equal>=10, therefore, the experiment of the present embodiment offer is successful.
If step 10 Success in Experiment, then calculating the content of drug resistance gene in testing sample, concrete grammar is as follows:
The computing formula of the content of m kind drug resistance gene isWherein, i is the i-th test zone of m kind drug resistance gene, n1 is the number of the test zone of m kind drug resistance gene, bi is the quantity of the order-checking fragment of the i-th test zone of m kind drug resistance gene, k is the endogenous standard gene of kth kind, and n3 is the number of endogenous standard gene, and j is the jth test zone of the endogenous standard gene of kth kind, n2 is the number of the test zone of the endogenous standard gene of kth kind, and aj is the quantity of the order-checking fragment of the jth kind test zone of the endogenous standard gene of kth kind; N is the sum of the test zone of all endogenous standard genes. The present invention adopts average to calculate the content of drug resistance gene, it is to reduce the impact on result of the factors such as amplification efficiency, if have detected the amplification efficiency of each multiplex amplification primer and ensureing that amplification efficiency is between 95%-105%, so amplification efficiency is little on the impact of result, therefore, it can the detection region only with a multiplex amplification primer amplification to represent drug resistance gene, endogenous standard gene and external source standard gene.
As can be seen from Table 1, in the present embodiment, have detected 4 kinds of drug resistance genes altogether, so m=4, each drug resistance gene have detected 1 test zone, so, n1=1, have detected 1 endogenous standard gene altogether, so n3=1, each endogenous standard gene have detected 1 test zone altogether, so, n2=1, N=1, table 1 lists the quantity of the order-checking fragment of each drug resistance gene and each endogenous standard gene, substitute them in drug resistance gene content computing formula in can obtain the content of each drug resistance gene, its result is in Table 1.
Step 11, judge whether testing sample contains drug resistance gene, and concrete grammar is as follows according to the content of drug resistance gene:
When judging method that testing sample contains drug resistance gene as the content>=�� 2 of: drug resistance gene, it is determined that testing sample contains drug resistance gene; When the content of all drug resistance genes is<during �� 2, it is determined that testing sample does not contain drug resistance gene; Wherein, �� 2 is decision threshold. �� 2 is that Stringency as requested is manually set, and when a small amount of drug resistance gene exists and namely may result in antibiotic ineffective, �� 2 value should be on the low side, on the contrary, and can be higher. When lacking amount and the effects of antibiotics relation of drug resistance gene (major part situation is such), �� 2 value can be set as 0.1%, in order between statistical error and sensitivity, obtain balance. In the present embodiment, �� 2 value is set as 0.1%. As can be seen from Table 1, in the present embodiment, except drug resistance gene CTX-M-14, the content of other three kinds of drug resistance genes is<�� 2=0.1% all, therefore, it is determined that testing sample is for being CTX-M-14 containing drug resistance gene, when the antibiotics of its correspondence prevents and treats rice disease, existence due to drug resistance, it is possible to poor effect. The present embodiment passes through 4 pairs of multiplex amplification primers, once the detection by quantitative content of 4 kinds of institute's drug resistance genes. On the basis of the present embodiment, it is only necessary to more multiplex amplification primer is set, the content of any multiple drug resistance gene wishing detection of detection by quantitative can be realized.
The testing result of checking the present embodiment, concrete grammar is as follows:
Multiplex amplification primer in synthetic table 1, the DNA utilizing each multiplex amplification primer pair testing sample one by one carries out pcr amplification, amplified production is carried out sepharose electrophoresis detection, find that drug resistance gene CTX-M-14 exists amplified band, it is judged as the positive, simultaneously, endogenous standard gene and external source standard gene all have obvious amplified band, it is judged to the positive, the amplified production of the primer of other three kinds of drug resistance genes is without obvious band, being judged to feminine gender, this result is consistent with the testing result of the present embodiment.
Embodiment two, Semen Maydis microorganism drug resistance gene detection
The maize leaf used in the present embodiment takes from the corn field in Zhuankou development zone, Wuhan, and the drug resistance gene detecting microorganism therein is in order to pointedly to maize diseases medication, it is to avoid the generation of drug resistance, meanwhile, reduces unnecessary medication, saves cost. The present embodiment comprises the steps:
By the method identical with examples of implementation one, it is determined that testing sample needs the external source standard gene of the endogenous standard gene in the drug resistance gene of microorganism of detection, testing sample and testing sample; Preparation is for expanding the multiplex amplification primer of the test zone of drug resistance gene, endogenous standard gene and external source standard gene; Result is in Table 2.
Table 2 is detected gene-correlation information and testing result in the present embodiment
By the method similar with examples of implementation one, in testing sample, add the exogenous nucleic acid that multiplex amplification primer can not expand, the genome of exogenous nucleic acid Yu testing sample is together extracted, obtains biased sample; Extract the genome of biased sample, it is thus achieved that the genomic total amount of biased sample be 3520ng; External source standard gene is added in the genome of biased sample, the ratio of the genomic gross mass of the quality of external source standard gene and biased sample is 1/1000, owing to the genomic total amount of biased sample is 3520ng, therefore, need to add 3.52ng external source standard gene, it is thus achieved that mixing nucleic acid.
By the method identical with examples of implementation one, utilize multiplex amplification primer pair mixing nucleic acid to expand, obtain amplified production, utilize amplified production to build high-throughput sequencing library; High-throughput sequencing library is carried out high-flux sequence, obtains order-checking fragment group; The quantity of the quantity of the order-checking fragment according to external source standard gene and the order-checking fragment of endogenous standard gene, it is judged that the experiment that the present embodiment provides is successful; Calculating the content of drug resistance gene in testing sample, result is in Table 2.
By the method identical with examples of implementation one, it is judged that in the present embodiment, except drug resistance gene CTX-M-14, the content of other three kinds of drug resistance genes all < �� 2=0.1%, therefore, it is determined that testing sample is for being CTX-M-14 containing drug resistance gene. By the method identical with examples of implementation one, the testing result of checking the present embodiment is correct.
The method that the embodiment of the present invention provides can detect different testing samples, highly versatile, and main difference is in that to need the difference according to testing sample, selects corresponding Extraction Methods of Genome to carry out genomic extraction. Order-checking is the ultimate standard of detection of nucleic acids, and therefore embodiment of the present invention testing result accuracy is high; Order-checking can distinguish single base difference, and therefore the resolution of embodiment of the present invention drug resistance gene detection is high. The method that the embodiment of the present invention provides can detect under need not cultivating the premise with purification, and therefore, detection is quick, and this is particularly significant for diagnosis and the timely medication in time of the state of an illness, and meanwhile, the detection for cultivating pathogen provides possibility. The embodiment of the present invention can detect any several drug resistance gene, and the antibiotic for finding a kind of effective prevention or treatment plant disease provides comprehensive information. Therefore, the embodiment of the present invention can be disposable, quantitative, quickly, accurately detection any amount and type microorganism in any multiple wish detection drug resistance gene, its effect is that prior art does not reach.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.
Claims (10)
1. the detection method of a phytomicroorganism drug resistance gene, it is characterised in that described method includes:
Determining the external source standard gene of the endogenous standard gene and described testing sample needed in testing sample in the drug resistance gene of microorganism of detection, described testing sample, described testing sample is whole plant plant or the described plant of part;
Preparation is for expanding the multiplex amplification primer of the test zone of described drug resistance gene, described endogenous standard gene and described external source standard gene;
Extract the genome of described testing sample;
Described external source standard gene is added, it is thus achieved that mixing nucleic acid in the genome of described testing sample;
Utilize mixing nucleic acid described in described multiplex amplification primer pair to expand, obtain amplified production, utilize described amplified production to build high-throughput sequencing library;
Described high-throughput sequencing library is carried out high-flux sequence, obtains order-checking fragment group;
Analyze described order-checking fragment group, it is thus achieved that the quantity of the order-checking fragment of the quantity of the order-checking fragment of drug resistance gene described in described testing sample, the quantity of order-checking fragment of described endogenous standard gene and described external source standard gene;
The quantity of the quantity of the order-checking fragment according to described external source standard gene and the order-checking fragment of described endogenous standard gene, it is judged that whether experiment is successful;
If described Success in Experiment, then calculate the content of drug resistance gene described in described testing sample;
Content according to described drug resistance gene judges whether described testing sample contains drug resistance gene.
2. detection method according to claim 1, it is characterised in that described drug resistance gene is at least 1, described endogenous standard gene is at least 1, and described external source standard gene is at least 1.
3. detection method according to claim 1, it is characterised in that described endogenous standard gene is the gene in the microorganism of described testing sample.
4. detection method according to claim 1, it is characterised in that described external source standard gene is not present in the biology of known group.
5. detection method according to claim 1, it is characterised in that be used for the homology expanding biological other region genomic in the design section of the primer of described drug resistance gene or amplification region and described testing sample lower than 98%.
6. detection method according to claim 1, it is characterized in that, described judgment experiment whether successfully method is: when the quantity of the quantity of order-checking fragment of described external source standard gene and the order-checking fragment of described endogenous standard gene equal>=�� 1 time, then Success in Experiment; When the quantity of order-checking fragment of described external source standard gene or the order-checking fragment of described endogenous standard gene quantity<during �� 1, then the failure of an experiment; Wherein, �� 1 is decision threshold.
7. detection method according to claim 1, it is characterised in that the method for the content of drug resistance gene described in described testing sample that calculates is: described in m kind, the computing formula of the content of drug resistance gene isWherein, i is the i-th test zone of drug resistance gene described in m kind, n1 is the number of the test zone of drug resistance gene described in described m kind, bi is the quantity of the order-checking fragment of the i-th test zone of described m kind drug resistance gene, k is endogenous standard gene described in kth kind, n3 is the number of described endogenous standard gene, j is the jth test zone of endogenous standard gene described in kth kind, n2 is the number of the test zone of endogenous standard gene described in kth kind, and aj is the quantity of the order-checking fragment of the jth kind test zone of endogenous standard gene described in kth kind; N is the sum of the test zone of all described endogenous standard genes.
8. detection method according to claim 1, it is characterised in that when judging the method whether containing described drug resistance gene in described testing sample as the content>=�� 2 of: described drug resistance gene, it is determined that described testing sample contains described drug resistance gene; When the content of all described drug resistance genes is<during �� 2, it is determined that described testing sample is without there being described drug resistance gene; Wherein, �� 2 is decision threshold.
9. detection method according to claim 1, it is characterised in that described method also includes:
In described testing sample, add the exogenous nucleic acid that described multiplex amplification primer can not expand, the genome of described exogenous nucleic acid Yu described testing sample is together extracted.
10. detection method according to claim 1, it is characterised in that the ratio of the quality of described external source standard gene and the genomic gross mass of described testing sample is more than 1/100000.
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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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