CN105567832A - Microorganism drug resistance gene detection method - Google Patents

Microorganism drug resistance gene detection method Download PDF

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CN105567832A
CN105567832A CN201610061081.5A CN201610061081A CN105567832A CN 105567832 A CN105567832 A CN 105567832A CN 201610061081 A CN201610061081 A CN 201610061081A CN 105567832 A CN105567832 A CN 105567832A
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drug resistance
gene
resistance gene
standard gene
testing sample
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CN105567832B (en
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彭海
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Jianghan University
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Abstract

The invention discloses a microorganism drug resistance gene detection method. The method includes the steps that a drug resistance gene, an endogenous standard gene and an exogenous standard gene are determined; multiple amplification primers are prepared; exogenous nucleic acid is added into a sample to be detected to obtain a mixed sample and extract a genome; the exogenous standard gene is added into the genome to obtain mixed nucleic acid; the mixed nucleic acid is amplified, a high-throughput sequencing library is constructed through amplification productimers, and high-throughput sequencing is conducted to obtain a sequencing fragment set; the sequencing fragment set is analyzed, and whether an experiment succeeds or not is judged according to the number of sequencing fragments of the obtained exogenous standard gene and the endogenous standard gene; if the experiment succeeds, the content of the drug resistance gene is calculated; according to the content of the drug resistance gene, whether the sample to be detected contains the drug resistance gene or not is judged. By means of the method, various drug resistance genes needing to be detected in any microorganism can be detected quantitatively at a time. Besides, microorganisms do not need to be cultured or purified during detection, the speed is high, and results are accurate and reliable.

Description

A kind of detection method of microorganism drug resistance gene
Technical field
The present invention relates to biological technical field, particularly a kind of detection method of microorganism drug resistance gene.
Background technology
Antibiotics is widely used in preventing and treating the vegeto-animal disease caused by microorganism, abuse of antibiotics class medicine can cause microbial evolution to go out drug resistance gene, and produce resistance, this makes antibiotics lose efficacy when disease preventing and treating, for effectively preventing and treating the disease caused by microorganism, avoid the abuse of antibiotics, drug resistance gene detection need be carried out to testing sample.
The method of existing detection drug resistance gene comprises: the pathogenic bacteria estimating the drug resistance gene of the needs detection that testing sample carries, be separated and this pathogenic bacteria of purifying, by PCR (PolymeaseChainReaction, polymerase chain reaction) this drug resistance gene of primer amplification, utilize electrophoresis or real time quantitative PCR method to judge whether to exist in testing sample the drug resistance gene needing to detect one by one.
Realizing in process of the present invention, contriver finds that prior art at least exists the one in following problem:
Need to cultivate, abstraction and purification pathogenic bacteria, make time of detecting longer, delay treatment opportunity; The drug resistance gene in not educable resistant organism cannot be detected; Once can only detect for a kind of or a few pathogenic bacteria; Once can only detect one or a few drug resistance gene, drug resistance genes all in testing sample can not be detected, the comprehensive information needed for medication can not be given; The detection by quantitative of drug resistance gene can not be realized.
Summary of the invention
In order to solve in prior art the problem that the method that detects drug resistance gene has much room for improvement, embodiments provide a kind of detection method of microorganism drug resistance gene.Described technical scheme is as follows:
Embodiments provide a kind of detection method of microorganism drug resistance gene, described method comprises:
Determine to need in testing sample the external source standard gene of endogenous standard gene in the drug resistance gene of the microorganism detected, described testing sample and described testing sample;
For the preparation of the multiplex amplification primer of the test zone of amplification described drug resistance gene, described endogenous standard gene and described external source standard gene;
Extract the genome of described testing sample;
In the genome of described testing sample, add described external source standard gene, obtain mixing nucleic acid;
Utilize and mix nucleic acid described in described multiplex amplification primer pair and increase, obtain amplified production, utilize described amplified production to build high-throughput sequencing library;
High-flux sequence is carried out to described high-throughput sequencing library, obtains sequenced fragments group;
Analyze described sequenced fragments group, obtain the quantity of the sequenced fragments of drug resistance gene described in described testing sample, the quantity of sequenced fragments of described endogenous standard gene and the quantity of the sequenced fragments of described external source standard gene;
According to the quantity of the quantity of the sequenced fragments of described external source standard gene and the sequenced fragments of described endogenous standard gene, judgment experiment whether success;
If described Success in Experiment, then calculate the content of drug resistance gene described in described testing sample;
Judge that whether described testing sample is containing drug resistance gene according to the content of described drug resistance gene.
Particularly, 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.
Particularly, described endogenous standard gene is the gene in the microorganism of described testing sample.
Particularly, described external source standard gene is not present in the biology of known group.
Particularly, other region genomic of the biology in the design section of the primer of the described drug resistance gene that increases or amplification region and described testing sample the homology in other region on zoic genome except drug resistance gene lower than 98%.
Particularly, described judgment experiment whether successfully method is: when the quantity of the sequenced fragments of described external source standard gene and the sequenced fragments of described endogenous standard gene quantity all >=α 1 time, then Success in Experiment; As the quantity < α 1 of the quantity of the sequenced fragments of described external source standard gene or the sequenced fragments of described endogenous standard gene, then the failure of an experiment; Wherein, α 1 is decision threshold.
Particularly, the method calculating the content of drug resistance gene described in described testing sample for: described in m kind, the calculation formula of the content of drug resistance gene is wherein, i-th test zone that i is 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 sequenced fragments of 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, the jth test zone that j is endogenous standard gene described in kth kind, n2 is the quantity of the number of the test zone of endogenous standard gene described in kth kind, the sequenced fragments of the jth kind test zone that aj is endogenous standard gene described in kth kind; N is the sum of the test zone of all described endogenous standard genes.
Particularly, during the content >=α 2 of the method whether containing described drug resistance gene of judging in described testing sample as: described drug resistance gene, judge that described testing sample contains described drug resistance gene; As the content < α 2 of all described drug resistance genes, judge described testing sample not containing described drug resistance gene; Wherein, α 2 is decision threshold.
Particularly, described method also comprises: in described testing sample, add the exogenous nucleic acid that described multiplex amplification primer can not increase, together extracted by the genome of described exogenous nucleic acid and described testing sample.
Particularly, the ratio of the quality of described external source standard gene and the genomic total mass of described biased sample is greater than 1/100000.
The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is: the method that the embodiment of the present invention provides can detect different testing samples, highly versatile, main difference point is to need the difference according to testing sample, select corresponding Extraction Methods of Genome, simultaneously, the method that the embodiment of the present invention provides can under not needing to cultivate the prerequisite with purifying, disposable, quantitative, fast, any multiple drug resistance gene of wishing to detect detected accurately in the microorganism of any amount and type, the comprehensive information needed for medication can be given.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, below embodiment of the present invention is described further in detail.
The drug resistance gene of embodiment one human excrement and urine microorganism detects
The kind of human excrement and urine's microbial reaction human intestinal pathogenic micro-organism and quantity are the foundations of enteron aisle class disease diagnosis and therapy.The human excrement and urine used in the present embodiment takes from the patient that diagnosis has enteritis, and the drug resistance gene detecting the microorganism in its ight soil is that the enteritis in order to use antibiotics to treat this patient to doctor provides foundation.The present embodiment comprises the steps:
Endogenous standard gene in step 1, the drug resistance gene determining the microorganism needing detection in testing sample, testing sample and the external source standard gene of 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, β-lactamase, cephalosporinase, kantlex and Liu Suanyan NEOMYCIN SULPHATE in the present embodiment, wherein, drug resistance gene APH is-Ia anti-kantlex and Liu Suanyan NEOMYCIN SULPHATE simultaneously (3').In the present embodiment, endogenous standard gene is a kind, and particularly, endogenous standard gene is ribosome rRNA gene, and this ribosome rRNA gene to be present in most biology and to have conservative region.In the present embodiment, external source standard gene is a kind, particularly, external source standard gene is ERCC-00004 gene, when it carries out homology comparison on NCBI (http://www.ncbi.nlm.nih.gov), do not find that it exists homologous sequence at known organism with reference in genome, namely external source standard gene is not present in known biological genome.Table 1 is detected gene-correlation information and detected result in the present embodiment, title in Table 1 in drug resistance gene title and sequence numbering and antibiotic resistance database (CRAD:TheComprehensiveAntibioticResistanceDatabase, network address is http://arpcard.mcmaster.ca/) is consistent with sequence numbering.
Table 1 is detected gene-correlation information and detected result in the present embodiment
In table 1 "/" indicate without.
Step 2, the multiplex amplification primer of test zone for the preparation of amplification drug resistance gene, endogenous standard gene and external source standard gene, concrete grammar is as follows:
Select the conservative region design multiplex amplification primer of drug resistance gene; For the homology in other region genomic of the biology in the design section of the primer of the drug resistance gene that increases or amplification region and testing sample lower than 98%, this requirement ensure that the detection of drug resistance gene is by the interference in other region genomic of the biology in testing sample.Particularly, the design section of the primer of drug resistance gene is conservative between different variant, the identical primer of such guarantee can amplify the different variants of identical drug resistance gene, if the homology in other region genomic of the biology in the design section of the primer of drug resistance gene and testing sample is lower than 98%, then this primer can not increase other region, therefore, the detection of drug resistance gene can not be disturbed.Otherwise, require that the homology in other region genomic of the biology in the amplification region of primer of drug resistance gene and testing sample is lower than 98%, like this, the amplified production of acquisition can be distinguished mutually with drug resistance gene, can not disturb the detection of drug resistance gene equally.Meanwhile, in multiplex amplification primer the amplification efficiency of often pair of primer all between 95% ~ 105%.
Particularly, download the sequence of the drug resistance gene in table 1 in antibiotic resistance database, the numbering of the sequence of each drug resistance gene downloaded is in table 1.Homology comparison is utilized to obtain 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, so all zones of this sequence is as the design section of conservative region and multiplex amplification primer.The design section of the multiplex amplification primer obtained above is carried out homology comparison by NCBI and other region except drug resistance gene on institute zoic genome, reservation homology lower than 98% the design section of multiplex amplification primer.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: log in match Mo Feishier company multiple PCR primer Photographing On-line webpage https: //ampliseq.com/, select " DNAHotspotdesigns (single-pool) " at " Applicationtype " option.The design section of the multiplex amplification primer of acquisition 100 N are coupled together, forms an artificial reference genome, and select " Custom " in " Selectthegenomeyouwishtouse " option after, 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, obtain the multiplex amplification primer for the drug resistance gene that increases, endogenous standard gene and external source standard gene.Each heavy primer of multiplex amplification primer sequence is synthesized one by one by Sangon Biotech (Shanghai) Co., Ltd., and the template sequence to be increased 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 amplification efficiency of each multiplex amplification primer is detected according to the operational manual (PartNumber4376784Rev.E) of the StepOne real-time PCR of match Mo Feishier company of the U.S., only retain amplification efficiency 95% ~ 105% multiplex amplification primer, 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, the results are shown in Table 1.Multiplex amplification primer is matched Mo Feishier company by the U.S. and is synthesized and provide use with the form of mixing liquid.The present embodiment adopts the multiple PCR technique that provides of match Mo Feishier company of the U.S., its as many as 12000 test zones that can simultaneously increase, therefore, the present invention have the ability disposable detection existing be hopeful the drug resistance gene that detects.
Step 3, in testing sample, add the exogenous nucleic acid that multiplex amplification primer can not increase, together extracted by the genome of exogenous nucleic acid and testing sample, obtain biased sample, concrete grammar is as follows:
If genome content is higher in testing sample, if testing sample is various vivo biological tissue, then exogenous nucleic acid need not be added.If genomic content is lower in testing sample, if the testing sample in the present embodiment is human body fecal microorganism, genome extracts difficulty, after needing to add in testing sample the exogenous nucleic acid that multiplex amplification primer can not increase, conveniently can extract the genome in testing sample, generally, need the exogenous nucleic acid adding 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, such as, when extracting in a large number, add-on should increase to some extent, when ultramicron is extracted, can suitably reduce, under existing genome extractive technique level, generally minimumly 1ng must not be less than.Because multiplex amplification primer can not increase exogenous nucleic acid, therefore, the exogenous nucleic acid added does not affect 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 as shown in SEQ ID NO:13, and ERCC-00024 gene is synthesized by Sangon Biotech (Shanghai) Co., Ltd..In the present embodiment, in ight soil, genome content is lower, when faeces DNA test kit extraction genome is pressed in employing, mixes after the exogenous nucleic acid 1ug added in the testing sample of 400mg, namely obtain biased sample, the genomic normal extraction in biased sample can be ensured.
The genome of step 4, extraction biased sample, concrete grammar is as follows:
By faeces DNA test kit (production company: MP company of the U.S., product article No.: 116570200, product English name: FastDNASPINkitforfeces) process specifications extract the genome of biased sample, the genomic nucleic acids for biased sample extracted.Utilize the double-stranded DNA program in spectrophotometer (Quawell company of the U.S. produces, and model is Q5000), detect the genomic amount of the biased sample obtained.In the present embodiment, the genomic total amount of biased sample is 1334ng.
Step 5, in the genome of biased sample, add external source standard gene, the ratio of the quality of external source standard gene and the genomic total mass of biased sample is greater than 1/100000, and obtain mixing nucleic acid, concrete grammar is as follows:
In the present embodiment, the ratio of the quality of external source standard gene and the genomic total mass of biased sample is 1/1000, and the genomic total amount due to biased sample is 1334ng, therefore, needs to add 1.334ng external source standard gene, obtains mixing nucleic acid.
Step 6, utilize multiplex amplification primer pair mixing nucleic acid to increase, obtain amplified production, utilize amplified production to build high-throughput sequencing library, concrete grammar is as follows:
Library construction Kit 2.0 (match 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 comprises following reagent: 5 × IonAmpliSeq tMhiFiMix, FuPa reagent, transferring reagent, sequence measuring joints solution and DNA ligase.The method of library construction presses the operational manual " IonAmpliSeq of this library construction Kit tMlibraryPreparation " (publication number: MAN0006735, version: A.0) carry out.The amplification system of multiplex PCR is as follows: 5 × IonAmpliSeq tMthe mixing liquid 4 μ l of the multiplex amplification primer of HiFiMix4 μ l, preparation, mixing nucleic acid 10ng and without enzyme water 11 μ 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, concrete grammar is: in the amplified production of multiplex PCR, add 2 μ lFuPa reagent, after mixing, by following program reaction in PCR instrument: 50 DEG C, and 10 minutes; 55 DEG C, 10 minutes; 60 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixture a, and mixture a is containing the amplified production solution through phosphorylation.The amplified production of phosphorylation is connected upper sequence measuring joints, and concrete grammar is: in mixture a, add transferring reagent 4 μ l, sequence measuring joints solution 2 μ l and DNA ligase 2 μ l, after mixing, by following program reaction in PCR instrument: 22 DEG C, and 30 minutes; 72 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixed solution b.10 μ l are dissolved in without in enzyme water after utilizing the ethanol precipitation methods purifying mixed solution b of standard.American I nvitrigen company is utilized to produce dsDNAHSAssayKit (article No. is Q32852) also detects according to its specification sheets, after obtaining the mass concentration of mixed solution b, the mixed solution b after purifying is diluted to 15ng/ml, obtains the high-throughput sequencing library that concentration is about 100pM.
Step 7, carry out high-flux sequence to high-throughput sequencing library, obtain sequenced fragments group, concrete grammar is as follows:
Utilize the high-throughput sequencing library and test kit IonPITemplateOT2200Kitv2 (invirtrigen company of U.S. production that obtain, article No. is 4485146) check order before ePCR (EmulsionPCR, emulsion polymerization enzyme chain reaction) amplification, working method is undertaken by the operational manual of this test kit.(invirtrigen company of the U.S. produces to utilize ePCR product and test kit IonPISequencing200Kitv2, article No. is 4485149) on Proton bis-generation high-flux sequence instrument, carry out high-flux sequence, working method is undertaken by the operational manual of this test kit.In the present embodiment, high-flux sequence amount is set to 1M sequenced fragments (1M=,100 ten thousand), and order-checking length is set to 500cycle (circulation), after order-checking terminates, obtains sequenced fragments group.
Step 8, analysis sequenced fragments group, obtain the quantity of the quantity of sequenced fragments of drug resistance gene in testing sample, the quantity of the sequenced fragments of endogenous standard gene and the sequenced fragments of external source standard gene, concrete grammar is as follows:
According to the primer of sequenced fragments, utilize blastall (version2.2.26) software, by the optimum configurations of its acquiescence, by the drug resistance gene that the multiplex amplification primer pair in the comparison to table 1 of sequenced fragments group is answered, on the surveyed area of endogenous standard gene and external source standard gene, retain with the homology of surveyed area higher than 98% sequenced fragments (may be obtained by non-specific amplification with the sequenced fragments come from lower than 98%), they represent the quantity of the sequenced fragments of drug resistance gene in testing sample respectively, the quantity of the quantity of the sequenced fragments of endogenous standard gene and the sequenced fragments of external source standard gene, it the results are shown in Table 1.
Step 9, quantity according to the quantity of the sequenced fragments of external source standard gene and the sequenced fragments of endogenous standard gene, judgment experiment whether success, concrete grammar is as follows:
Judgment experiment whether successfully method is: when the quantity of the sequenced fragments of external source standard gene and the sequenced fragments of endogenous standard gene quantity all >=α 1, then Success in Experiment; As the quantity < α 1 of the quantity of the sequenced fragments of external source standard gene or the sequenced fragments of endogenous standard gene, then the failure of an experiment; Wherein, α 1 is decision threshold.If the amount of external source standard gene is too low, may be that high-throughput library construction or high-flux sequence are unsuccessful, if endogenous standard gene amount is too low, may is that nucleic acid extraction is unsuccessful, if test unsuccessful, adjusts in light of the circumstances, till Success in Experiment.In the present embodiment, α 1 gets 10 sequenced fragments, as can be seen from Table 1, the quantity of the quantity of the sequenced fragments of external source standard gene and the sequenced fragments of endogenous standard gene all >=10, therefore, the experiment that the present embodiment provides is successful.
If step 10 Success in Experiment, then calculate the content of drug resistance gene in testing sample, concrete grammar is as follows:
The calculation formula of the content of m kind drug resistance gene is wherein, i is 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 sequenced fragments of 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 a 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 sequenced fragments 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 mean number to calculate the content of drug resistance gene, to reduce the impact of the factors such as amplification efficiency on result, if have detected the amplification efficiency of each multiplex amplification primer and ensure that amplification efficiency is between 95%-105%, so the impact of amplification efficiency on result is little, therefore, can only adopt the surveyed area of 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 sequenced fragments of each drug resistance gene and each endogenous standard gene, and they are substituted into the content that can obtain each drug resistance gene in the calculation formula of the content of drug resistance gene, and it the results are shown in Table 1.
Step 11, judge that whether testing sample is containing drug resistance gene according to the content of drug resistance gene, concrete grammar is as follows:
When judging content >=α 2 as: drug resistance gene of method that testing sample contains drug resistance gene, judge that testing sample contains drug resistance gene; As the content < α 2 of all drug resistance genes, judge testing sample not containing drug resistance gene; Wherein, α 2 is decision threshold.α 2 is that Stringency as requested artificially sets, when a small amount of drug resistance gene exist can cause antibiotic ineffective time, α 2 value should be on the low side, on the contrary, can be higher.When lacking amount and the effects of antibiotics relation of drug resistance gene (most of situation is like this), α 2 value can be set as 0.1%, so that obtain balance between statistical error and sensitivity.In the present embodiment, α 2 value is set as 0.1%.As can be seen from Table 1, in the present embodiment, except drug resistance gene cepS, the content of other three kinds of drug resistance genes all >=α 2=0.1%, therefore, judge testing sample as containing drug resistance gene as CTX-M-14, blaNDM-1 and APH (3')-Ia, antibiotics corresponding to these three kinds of drug resistance genes by the existence due to resistance, causes poor effect for the treatment of patient.The present embodiment by 4 pairs of multiplex amplification primers, the detection by quantitative content of 4 kinds of institute's drug resistance genes once.On the basis of the present embodiment, only need to arrange more multiplex amplification primer, any multiple content of wishing the drug resistance gene detected of detection by quantitative can be realized.
The detected result of checking the present embodiment, concrete grammar is as follows:
Multiplex amplification primer in synthetic table 1, the DNA of each multiplex amplification primer pair testing sample is utilized to carry out pcr amplification one by one, agarose electrophoresis detection is carried out to amplified production, there is amplified band in discovery drug resistance gene CTX-M-14, blaNDM-1 and APH (3')-Ia, be judged as the positive, simultaneously, endogenous standard gene and external source standard gene all have obvious amplified band, be judged to be the positive, the amplified production of the primer of cepS is without obvious band, be judged to be feminine gender, this result is consistent with the detected result of the present embodiment.
Embodiment two: the detection of soil microorganisms drug resistance gene
Soil microorganisms derives from physical environment, and wherein drug resistance gene has reacted the situation that resistance element class medicine discharges in the environment to a certain extent.Testing sample in the present embodiment is soil, and this soil is taken from the rice terrace in Zhuankou development area, Wuhan, Hubei.
Identical drug resistance gene, endogenous standard gene and external source standard gene is determined by with embodiment one same procedure, and obtain identical multiplex amplification primer and surveyed area, adopt TIANGEN Biotech (Beijing) Co., Ltd. produce soil genome DNA extracting reagent kit (article No.: DP336) and by its operational manual carry out biased sample genome extract, the genomic total amount of the extraction of biased sample is 2350ng.After adding 2.350ng exogenous nucleic acid by the method identical with embodiment one, build high-throughput sequencing library and carry out high-flux sequence, obtain sequenced fragments group, after sequenced fragments group is analyzed, obtain the quantity of the quantity of sequenced fragments of drug resistance gene, the quantity of the sequenced fragments of endogenous standard gene and the sequenced fragments of external source standard gene, and calculating their content, it the results are shown in Table 2.By the method identical with embodiment one, setting α 2=0.1%, judge that the drug resistance gene that exists in testing sample is as CTX-M-14 and blaNDM-1, by the method that embodiment one is identical, carry out experimental verification, the result is identical with the detected result of the present embodiment.
Table 2 is detected gene-correlation information and detected result in the present embodiment
In table 2 "/" indicate without.
The method that the embodiment of the present invention provides can detect different testing samples, and highly versatile, main difference point is 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 the accuracy of embodiment of the present invention detected result 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 not need to cultivate with the prerequisite of purifying under detect, therefore, detection is fast, this timely diagnosis for the state of an illness and timely medication very important, meanwhile, for the detection can not cultivating pathogen provides possibility.The embodiment of the present invention can detect any several drug resistance gene, for the microbiotic finding a kind of effective prevention or disease therapy provides comprehensive information.Therefore, the embodiment of the present invention can be disposable, quantitative, fast, any multiple drug resistance gene of wishing to detect detected accurately in the microorganism of any amount and type, its effect is that prior art does not reach.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (10)

1. a detection method for microorganism drug resistance gene, is characterized in that, described method comprises:
Determine to need in testing sample the external source standard gene of endogenous standard gene in the drug resistance gene of the microorganism detected, described testing sample and described testing sample;
For the preparation of the multiplex amplification primer of the test zone of amplification described drug resistance gene, described endogenous standard gene and described external source standard gene;
Extract the genome of described testing sample;
In the genome of described testing sample, add described external source standard gene, obtain mixing nucleic acid;
Utilize and mix nucleic acid described in described multiplex amplification primer pair and increase, obtain amplified production, utilize described amplified production to build high-throughput sequencing library;
High-flux sequence is carried out to described high-throughput sequencing library, obtains sequenced fragments group;
Analyze described sequenced fragments group, obtain the quantity of the sequenced fragments of drug resistance gene described in described testing sample, the quantity of sequenced fragments of described endogenous standard gene and the quantity of the sequenced fragments of described external source standard gene;
According to the quantity of the quantity of the sequenced fragments of described external source standard gene and the sequenced fragments of described endogenous standard gene, judgment experiment whether success;
If described Success in Experiment, then calculate the content of drug resistance gene described in described testing sample;
Judge that whether described testing sample is containing drug resistance gene according to the content of described drug resistance gene.
2. detection method according to claim 1, is characterized in that, 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.
3. detection method according to claim 1, is characterized in that, described endogenous standard gene is the gene in the microorganism of described testing sample.
4. detection method according to claim 1, is characterized in that, described external source standard gene is not present in the biology of known group.
5. detection method according to claim 1, is characterized in that, for the homology in other region genomic of the biology in the design section of the primer of the described drug resistance gene that increases 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 sequenced fragments of described external source standard gene and the sequenced fragments of described endogenous standard gene quantity all >=α 1 time, then Success in Experiment; As the quantity < α 1 of the quantity of the sequenced fragments of described external source standard gene or the sequenced fragments of described endogenous standard gene, then the failure of an experiment; Wherein, α 1 is decision threshold.
7. detection method according to claim 1, is characterized in that, the method calculating the content of drug resistance gene described in described testing sample for: described in m kind, the calculation formula of the content of drug resistance gene is wherein, i-th test zone that i is 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 sequenced fragments of 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, the jth test zone that j is endogenous standard gene described in kth kind, n2 is the quantity of the number of the test zone of endogenous standard gene described in kth kind, the sequenced fragments of the jth kind test zone that aj is 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, is characterized in that, during the content >=α 2 of the method whether containing described drug resistance gene of judging in described testing sample as: described drug resistance gene, judges that described testing sample contains described drug resistance gene; As the content < α 2 of all described drug resistance genes, judge described testing sample not containing described drug resistance gene; Wherein, α 2 is decision threshold.
9. detection method according to claim 1, is characterized in that, described method also comprises:
In described testing sample, add the exogenous nucleic acid that described multiplex amplification primer can not increase, the genome of described exogenous nucleic acid and described testing sample is together extracted.
10. detection method according to claim 1, is characterized in that, the ratio of the quality of described external source standard gene and the genomic total mass of described biased sample is greater than 1/100000.
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