CN105255998A - Method for fast searching pathogen specific detection target - Google Patents
Method for fast searching pathogen specific detection target Download PDFInfo
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- CN105255998A CN105255998A CN201510341295.3A CN201510341295A CN105255998A CN 105255998 A CN105255998 A CN 105255998A CN 201510341295 A CN201510341295 A CN 201510341295A CN 105255998 A CN105255998 A CN 105255998A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Abstract
The invention discloses a method for fast searching a pathogen specific detection target. The method realizes fast searching of a species-specific gene of a novel pathogen in a genome level and building of novel pathogen specific molecule diagnosis in short time, and is suitable for fast detection and diagnosis of sudden disease and novel variant deep-unstudied pathogens of which pathological features and propagation approaches are unknown.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to a kind of quick-searching pathogen specific detection target calibration method.
Background technology
Pathogen is the general designation of the life entity that can cause plant or animal diseases.But the diagnostic method at present for causal organism is single, efficiency is low, specificity is poor.So, the screening of setting up the specific detection target of the pathogen that causal organism especially newly makes a variation be fast, the basis of the Diagnosis and treatment of effective implemention pathogen.
Detection for pebrine disease can trace back to 1870, and pasteur has been founded female moth Microscopical Method For Detection and detected From Nosema bombycis, and over more than 100 year, this method is used till today always, is the traditional method for detecting on producing.But the method is comparatively complicated, comparatively strong for operator's skill requirement, and different people operation to there is error larger.Therefore, at present for the detect delay focus of this disease mainly concentrate on find one more simply, detection method faster.Up to the present the detection of nosema bombycis experienced by following several stages: microscope inspection, molecular Biological Detection, immunology detection.In recent years, progressively there have been developed a series of immunological detection methods that it is carried out for the antigenic difference of sporoderm protein between microsporidium kind.But, because the establishment test period of immunological detection method is longer, Financial cost is comparatively large, therefore, constrain applying of this method to a certain extent.But, along with the development of Protocols in Molecular Biology, within 2004, nosema bombycis genome sequencing completes, and depicts frame diagram, the detection of pebrine starts to enter the molecular biology stage, utilizes the method for round pcr or LAMP technology detection of particles disease to begin to appear in the newspapers.There is shorter, the plurality of advantages such as input cost is low research cycle in PCR detection kit as early as possible, but, owing to there is no a kind of unified target gene determination way in prior art, therefore, there is the poor problem of sensitivity, specificity in PCR detection kit existing in the market.So, set up as early as possible a species specificity better, target gene search method that specific aim is stronger just becomes the most important thing of pebrine testing.
Summary of the invention
An object of the present invention is for solving at present for the difficult problem that diagnostic method is single, efficiency is low, specificity is poor of causal organism, providing a kind of quick-searching pathogen specific detection target calibration method.
The invention provides a kind of quick-searching pathogen specific detection target calibration method, comprise the following steps:
Gather the sample of novel causal organism, extract its genome, build genomic library;
By the announced genomic information of NCBI, based on the conservative property of housekeeping gene loci, carry out karyomit(e) or scaffold aligns one by one, carry out Collinearity Diagnosis Analysis simultaneously;
Retrieve the gene that locus is not guarded, and determine the gene order alternatively gene in itself and GenBank storehouse without homology by Comparative genomic strategy Epidemiological Analysis;
Candidate gene is retrieved in object pathogen genomic data, determines its upstream and downstream gene conservative property, and build storehouse separately;
Candidate gene is cloned, checks order, compare of analysis, and detect the expression of gene;
To verify correct, and the gene of normal transcription can be defined as the target gene of molecular diagnosis;
Collect the pathogenic micro-organism with same host that variform is different, and candidate gene is carried out PCR detection, verify its specificity.
Further, the target gene that specificity is good is made PCR quick detection kit, or makes immunity detection reagent.
Beneficial effect of the present invention is: quick-searching pathogen specific detection target calibration method of the present invention is at the species specific gene of the novel causal organism of genomic level fast finding, the specific molecular diagnosis of novel pathogen can be set up in the short period of time, be applicable to rapid detection and the diagnosis of the not deep pathogen of people's researchs such as pathological characteristics such as burst epidemic disease, novel variant pathogen etc. is unknown, route of transmission is unknown.
Accompanying drawing explanation
Figure 1 shows that the Collinearity Diagnosis Analysis figure in the specific gene group region, one end in the embodiment of the present invention on No. 32 Scaffold.
Figure 2 shows that the RT-PCR proof diagram of NbPolk and NbOPI two encoding genes in the embodiment of the present invention.
Figure 3 shows that the PCR detection figure of six kinds of silkworm pathogenic micro-organism strain isolateds in the embodiment of the present invention.
Embodiment
Hereafter will describe the specific embodiment of the invention in detail in conjunction with concrete accompanying drawing.It should be noted that the combination of technical characteristic or the technical characteristic described in following embodiment should not be considered to isolated, they can mutually be combined thus be reached better technique effect.
Embodiment
Quick-searching pathogen specific detection target calibration method of the present invention, comprises the following steps:
Gather the sample of novel causal organism, extract its genome, build genomic library;
By the announced genomic information of NCBI, based on the conservative property of housekeeping gene loci, carry out karyomit(e) or scaffold aligns one by one, carry out Collinearity Diagnosis Analysis simultaneously;
Retrieve the gene that locus is not guarded, and determine the gene order alternatively gene in itself and GenBank storehouse without homology by Comparative genomic strategy Epidemiological Analysis;
Candidate gene is retrieved in object pathogen genomic data, determines its upstream and downstream gene conservative property, and build storehouse separately;
Candidate gene is cloned, checks order, compare of analysis, and detect the expression of gene;
To verify correct, and the gene of normal transcription can be defined as the target gene of molecular diagnosis;
Collect the pathogenic micro-organism with same host that variform is different, and candidate gene is carried out PCR detection, verify its specificity.
Further, the target gene that specificity is good is made PCR quick detection kit, or makes immunity detection reagent.
At present, this invention has been applied on the Pathogen test of pebrine disease.
Concrete implementation example:
Be separated the pebrine disease silkworm gathering Chongqing region, extract its totalRNA, and carry out genome sequencing.After this, to nosema bombycis (NosemabombycisCQ1) full-length genome 4, 479 genes compare genomics analysis, by with other other microsporidium genomic informations announced, comprise Encephalitozoonromaleae, Encephalitozoonintestinalis, Encephalitozoonhellem, Encephalitozoonbieneusi, Nosemacerannae, Vittaformacorneae, Edhazardiaaedis, Antonosporalocustae, Vavraiaculicisfloridiensis, Trichomonashominis, Nematocidasp1 etc. are from people, cat, rabbit, honeybee, anopheles, be separated the microsporidium obtained in the hosts such as fruit bat to compare one by one, obtain 82 nosema bombycis Orphan genes (orphangene), for nosema bombycis is exclusive, and in GenBank, there is not homologous sequence and gene family information.
As shown in Figure 1, to the nearly edge species that NCBI can retrieve, in GenBank, download its genome sequence, left side is the microsporidium evolutionary relationship of six kinds of known group data, and right side is the special target gene of the pathogenic micro-organism of the infected silkworm obtained by Collinearity Diagnosis Analysis in case.Wherein each is classified as homologous gene and the seat in genome thereof, and horizontal line represents genome splicing situation and genetic interval distance.Candidate gene is retrieved in object pathogen genomic data, determines its upstream and downstream gene conservative property, and build storehouse separately; Codon preference shows, and these genes are inclined to use UCG (Ser) and CCG (Pro) two class codon.By investigating the GC distribution characteristics of these 82 gene place scaffold, result screens 4 for recently to insert gene, the display of GeneOntology (GO) functional classification is carried out to these four genes, they mainly participate in the functional gene of DNA reparation and apoptosis path, and this genoid is built storehouse separately.
Design the upstream and downstream special primer of candidate gene respectively, carry out T clone, and check order.By the gene of sequencing result matching degree >=98% alternatively.Meanwhile, with pebrine disease silkworm totalRNA for template carries out the expression analysis of candidate gene, and determine that it transcribes period, by the gene alternatively target gene of susceptible middle and later periods stably express.
As shown in Figure 2, extract disease silkworm larva phase 1-10 days respectively, adult stage is (because it is development by metamorphosis, therefore get pupa, moth, ovum period respectively) totalRNA, the special primer analyzing the candidate gene obtained before getting carries out transcription analysis, and result shows, respectively compared with the contrast of silkworm, pathogen, these two genes are lasting, stable all after infection transcribes, and is suitable as candidate targets gene.Mate completely by sequence verification, therefore by arduous separately for candidate gene correct for checking, and be defined as the target gene of molecular diagnosis.
As shown in Figure 3, select the different sick silkworm of trouble 6 kinds to be template, utilize the target gene special primer in candidate gene storehouse to carry out pcr amplification, result display GENEI, geneII, gene IV specificity is best, can assemble test kit.
At present, carried out the preparation of pebrine disease LAMP detection kit, wherein GeneI and GeneIV specificity is better, and sensitivity reaches 1 × 10
5individual pathogen.
The present invention is based on the pathogenic micro-organism to a kind of infected silkworm---nosema bombycis is the research method that a kind of that research object obtains promotes, applicability is strong.
In addition, after this method is established, the rapid detection for the pathogenic micro-organism solving unknown pathogen or the variation fast having completed gene order-checking provides a kind of effective method.
Quick-searching pathogen specific detection target calibration method of the present invention is at the species specific gene of the novel causal organism of genomic level fast finding, the molecular diagnosis specifically of novel pathogen can be set up in the short period of time, be applicable to rapid detection and the diagnosis of the not deep pathogen of people's researchs such as pathological characteristics such as burst epidemic disease, novel variant pathogen etc. is unknown, route of transmission is unknown.
Although give some embodiments of the present invention, it will be understood by those of skill in the art that without departing from the spirit of the invention herein, can change embodiment herein.Above-described embodiment is exemplary, should using embodiment herein as the restriction of interest field of the present invention.
Claims (2)
1. a quick-searching pathogen specific detection target calibration method, is characterized in that, comprise the following steps:
Gather the sample of novel causal organism, extract its genome, build genomic library;
By the announced genomic information of NCBI, based on the conservative property of housekeeping gene loci, carry out karyomit(e) or scaffold aligns one by one, carry out Collinearity Diagnosis Analysis simultaneously;
Retrieve the gene that locus is not guarded, and determine the gene order alternatively gene in itself and GenBank storehouse without homology by Comparative genomic strategy Epidemiological Analysis;
Candidate gene is retrieved in object pathogen genomic data, determines its upstream and downstream gene conservative property, and build storehouse separately;
Candidate gene is cloned, checks order, compare of analysis, and detect the expression of gene;
To verify correct, and the gene of normal transcription can be defined as the target gene of molecular diagnosis;
Collect the pathogenic micro-organism with same host that variform is different, and candidate gene is carried out PCR detection, verify its specificity.
2. quick-searching pathogen specific detection target calibration method as claimed in claim 1, is characterized in that, also comprise step target gene being made PCR quick detection kit or immunity detection reagent.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111020018A (en) * | 2019-11-28 | 2020-04-17 | 天津金匙医学科技有限公司 | Macrogenomics-based pathogenic microorganism detection method and kit |
| CN112863601A (en) * | 2021-01-15 | 2021-05-28 | 广州微远基因科技有限公司 | Pathogenic microorganism drug-resistant gene attribution model and establishing method and application thereof |
Citations (2)
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| EP0837142A1 (en) * | 1996-10-15 | 1998-04-22 | Smithkline Beecham Corporation | Method for determining gene essentiality in a pathogen |
| CN101501199A (en) * | 2006-02-10 | 2009-08-05 | 孟山都技术有限公司 | Identification and use of target genes for control of plant parasitic nematodes |
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2015
- 2015-06-18 CN CN201510341295.3A patent/CN105255998A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0837142A1 (en) * | 1996-10-15 | 1998-04-22 | Smithkline Beecham Corporation | Method for determining gene essentiality in a pathogen |
| CN101501199A (en) * | 2006-02-10 | 2009-08-05 | 孟山都技术有限公司 | Identification and use of target genes for control of plant parasitic nematodes |
Non-Patent Citations (2)
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| 罗洁: "Nosema属微孢子虫基因组特异共线性区域的进化与功能分析", 《中国博士学位论文全文数据库 基础科学辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111020018A (en) * | 2019-11-28 | 2020-04-17 | 天津金匙医学科技有限公司 | Macrogenomics-based pathogenic microorganism detection method and kit |
| CN112863601A (en) * | 2021-01-15 | 2021-05-28 | 广州微远基因科技有限公司 | Pathogenic microorganism drug-resistant gene attribution model and establishing method and application thereof |
| CN112863601B (en) * | 2021-01-15 | 2023-03-10 | 广州微远基因科技有限公司 | Pathogenic microorganism drug-resistant gene attribution model and establishing method and application thereof |
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