CN101705300B - Special probe and gene chip used for identifying penicillium marneffei - Google Patents
Special probe and gene chip used for identifying penicillium marneffei Download PDFInfo
- Publication number
- CN101705300B CN101705300B CN2009102372277A CN200910237227A CN101705300B CN 101705300 B CN101705300 B CN 101705300B CN 2009102372277 A CN2009102372277 A CN 2009102372277A CN 200910237227 A CN200910237227 A CN 200910237227A CN 101705300 B CN101705300 B CN 101705300B
- Authority
- CN
- China
- Prior art keywords
- sequence
- penicillium marneffei
- dna
- probe
- gene chip
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a special probe and gene chip used for identifying penicillium marneffei. The special probe for identifying penicillium marneffei provided by the invention comprises at least one of the following eight DNA fragments: four DNA fragments of which nucleotide sequences are from sequence 1 to sequence 4 in the sequence table and four DNA fragments of which nucleotide sequences are complementary sequences of the sequence 1 to sequence 4 in the sequence table. The invention also provides a DNA chip containing the special probe for identifying penicillium marneffei. The special probe, DNA chip or device is all used for identifying penicillium marneffei. When in use, the special probe is coated on a low-density gene chip membrane, then the flow-through hybridization between the ITS region of rDNA of the strain to be tested and the species-specific probe on the low-density gene chip membrane is carried out by using the test platform based on the flow-through hybridization technology, and then color development is carried to finally identify the penicillium marneffei. The special probe and gene chip of the invention are characterized by strong specificity and fast operation and can play fast, specific and positive role in testing the penicillium marneffei in clinical specimens.
Description
Technical field
The present invention relates to a kind of application specific probe and gene chip of identifying the Penicillium marneffei bacterium.
Background technology
Along with being on the increase of AIDS (AIDS) patient; The morbidity of opportunistic fungal infection is also in increase at full speed, and the aggressive penicilliosis that is wherein caused by Penicillium marneffei has become south east asia and China Guangdong, area, Guangxi AIDS patient's significant opportunistic fungal infection.Identify this important pathogen rapidly and accurately, have crucial meaning for China AIDS patient's integrated control.
The inspection means that tradition is used for the infection of Penicillium marneffei bacterium mainly comprise fungus microscope examination, cultivation, evaluation and antifungal drug sensitivity testing etc., but these methods often take length, approximately need 3-4 week; And the positive rate that detects is low.Fast, responsive, detect the Penicillium marneffei bacterium specifically, become the major issue that presses for solution clinically, the solution of this problem is for the responsive medicine of correct diagnosis, choose reasonable and formulate regimen and have crucial meaning.
During technology that from clinical samples, directly detects fungi that molecular biology is relevant and method are constantly developing; The most active round pcr than methods such as fungi cultivation have fast, characteristics such as sensitivity, this technology fungi composition in the samples such as serum, blood plasma, whole blood, urine, sputum, BAL fluid and fester that can increase.But, still can not detect the Penicillium marneffei bacterium in the clinical samples fast, specifically at present.
Water conservancy diversion hybridization technique (Flow-through hybridization; The US patent No. 6020187 and 5741647) be with probe stationary on the matter matrix; When test sample is flowed through the matter matrix that places the water conservancy diversion hybrid device; Target sequence and probe hybridization form complex body, reflect the nucleic acid target molecule in the sample to be detected thereby detect hybridization complex through colour developing then.The hybridizing method that this technology is more traditional has high specificity, characteristics fast; If can this technology and high-throughput low density gene chip technology and the high round pcr of susceptibility be combined, then can be the Penicillium marneffei bacterium in detecting clinical samples.
Summary of the invention
The purpose of this invention is to provide a kind of application specific probe and gene chip of identifying the Penicillium marneffei bacterium.
The invention provides the application specific probe of a kind of evaluation Penicillium marneffei bacterium (Penicillium marneffei), be made up of in following 8 dna fragmentations at least one: nucleotide sequence is respectively 4 dna fragmentations of reverse complementary sequence of 4 dna fragmentations and sequence 1 to the sequence 4 that nucleotide sequence is sequence table respectively of sequence 1 to the sequence 4 of sequence table.More than 8 dna fragmentations; All be according to the design of the conserved sequence of Penicillium marneffei bacterium (Penicilliummarneffei); Wherein each dna fragmentation, or per two dna fragmentations, per three dna fragmentations, per four dna fragmentations ... Until the segmental combination of all DNA, all can be used for identifying Penicillium marneffei bacterium (Penicillium marneffei).
The present invention also protects the DNA chip that is used to identify Penicillium marneffei bacterium (Penicillium marneffei), contains above arbitrary described application specific probe.
The present invention protects the device of identifying Penicillium marneffei bacterium (Penicillium marneffei) simultaneously, comprises above arbitrary described application specific probe or above arbitrary described DNA chip.Said device can comprise that also the primer that Nucleotide is formed shown in the sequence 6 of sequence 5 and sequence table of sequence table is right.For the ease of detecting the results of hybridization of DNA to be measured and said probe, said primer on can be marked with vitamin H.
More than arbitrary described application specific probe, more than arbitrary described DNA chip or above arbitrary described device all can be applicable to identify Penicillium marneffei bacterium (Penicillium marneffei).
The present invention also protects the method for a kind of evaluation Penicillium marneffei bacterium (Penicillium marneffei); Be with the ITS district of the rDNA of bacterial strain to be measured respectively with said application specific probe hybridization; If the result is positive; Bacterial strain to be measured is Penicillium marneffei bacterium (Penicillium marneffei), if the result is negative, bacterial strain to be measured is non-Penicillium marneffei bacterium (Penicillium marneffei).Total DNA that the ITS district of the rDNA of said bacterial strain to be measured specifically can be with bacterial strain to be measured is a template, and pcr amplification obtains the primer of forming with Nucleotide shown in the sequence 6 of sequence of sequence table 5 and sequence table to carrying out.
The present invention also protects the method that whether contains Penicillium marneffei bacterium (Penicilliummarneffei) in a kind of detection sample, it is characterized in that: said method comprises the steps:
(1) the total DNA with sample to be tested is a template, and the primer of forming with Nucleotide shown in the sequence 6 of sequence of sequence table 5 and sequence table obtains target dna to carrying out pcr amplification; (2) target dna is hybridized with dna fragmentation shown in the sequence 4 (PM2) of sequence 1 (PM1) to the sequence table of sequence table respectively; Pass judgment on as follows according to results of hybridization: the results of hybridization like infructescence 1 and/or sequence 2 is positive, contains Penicillium marneffei bacterium (Penicillium marneffei) in the said sample; Results of hybridization like infructescence 3 and/or sequence 4 is positive, possibly contain the Penicillium marneffei bacterium in the said sample.
The present invention also protects the method that whether does not contain Penicillium marneffei bacterium (Penicilliummarneffei) in a kind of detection sample; It is characterized in that: said method comprises the steps: that (1) is template with total DNA of sample to be tested; The primer of forming with Nucleotide shown in the sequence 6 of sequence of sequence table 5 and sequence table obtains target dna to carrying out pcr amplification; (2) target dna is hybridized with dna fragmentation shown in the sequence 4 of sequence 1 to the sequence table of sequence table respectively; Pass judgment on as follows according to results of hybridization: the results of hybridization like infructescence 1 and/or sequence 2 is not positive, does not contain Penicillium marneffei bacterium (Penicillium marneffei) in the said sample; Results of hybridization like infructescence 3 (A-P-SPEC1) and/or sequence 4 (A-P-SPEC2) is not positive, does not contain the Penicillium marneffei bacterium in the said sample.
The present invention utilizes one of the most frequently used target sequence of PCR method detection fungi-Nei to distinguish (Internaltranscriptional space between transcribing; ITS); This is the tandem repetitive sequence of a multiple copied; Its 28s, 5.8s and 18s rDNA have conservative property between planting, so can be used to design universal primer, carry out pcr amplification then; And ITS1 and ITS2 district have mutability, therefore can carry out the selection of specific specificity probe.Among the present invention the specific specificity probe is coated on the low density gene chip film; Then above-mentioned PCR product is passed through the detection platform based on the water conservancy diversion hybridization technique; Carry out water conservancy diversion hybridization and colour developing with the specific specificity probe on the low density gene chip film, thereby finally realize the rapid detection of Penicillium marneffei bacterium in the clinical samples.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.
Dna fragmentation used in following examples is following:
Specific probe to the ITS district of the nucleic acid rDNA of Penicillium marneffei bacterium:
The Penicillium marneffei specific probe:
PM1 (Penicillium marneffei): 5 '-ACCGGGGCCACCCGGTCGCCG-3 '; The sequence 1 of sequence table.
PM2 (Penicillium marneffei): 5 '-TGTGAACCCTGATGAAGATGGA-3 '; The sequence 2 of sequence table.
A-P-SPEC1:5 '-TCCTCGAGCGTATGGGGCT-3 '; The sequence 3 of sequence table.
A-P-SPEC2:5 '-CGGCTTGTGTGTTGGG-3 '; The sequence 4 of sequence table.
Above-mentioned probe is coated on respectively on the gene chip film (nitrocellulose filter).
The primer in the ITS district of the rDNA of amplification Penicillium marneffei bacterium is to (the primer marked has vitamin H):
Forward:5 '-TCCGTAGGTGAACCTGCGGAAGGATCA-3 '; The sequence 5 of sequence table;
Reverse:5 '-CGCTTATTGATATGCTTAAGTTCAGCG-3 '; The sequence 6 of sequence table.
Penicillium marneffei bacterium sample used in following examples is available from Peking University fungi and mycosis research centre DSMZ, and the numbering of each bacterial classification sample is following:
Penicillium marneffei bacterium (Penicillium marneffei): BMU3156.
The pathogenic fungus of non-Penicillium marneffei bacterium:
Candida albicans (Candida albicans): ATCC24433;
Candida parapsilosis (Candida parapsilosis): ATCC22019;
The detection of embodiment 1, Penicillium marneffei bacterium
The sample that will contain the Penicillium marneffei bacterium respectively extracts genomic dna according to standard method; Use above-mentioned primer that (sequence 5 and the sequence 6 of sequence table) carried out PCR reaction (about 1.5 hours) then; Afterwards PCR product and the nitrocellulose filter that is marked with different probe (being respectively probe shown in sequence 1 to the sequence 4) are carried out the water conservancy diversion hybridization through medical making nucleic acid molecular hybridization appearance; Result according to reaction judges the bacterial classification in the sample to be detected, and required time is 3.5 hours.The result sees table 1.
The qualification result of table 1 Penicillium marneffei bacterium
| Sample | Detected result |
| Penicillium marneffei bacterium (Penicillium marneffei) | PM1, the positive A-P-SPEC1 of PM2, the A-P-SPEC2 positive |
| Candida albicans (Candida albicans) | PM1, the negative A-P-SPEC1 of PM2, A-P-SPEC2 feminine gender |
| Candida parapsilosis (Candida parapsilosis) | PM1, the negative A-P-SPEC1 of PM2, A-P-SPEC2 feminine gender |
The detection of Penicillium marneffei bacterium in embodiment 2, the mixed bacterium
The sample of mixed bacterium that will contain the pathogenic fungi of Penicillium marneffei bacterium and other kind extracts genomic dna according to standard method; Use above-mentioned primer that (sequence 5 and the sequence 6 of sequence table) carried out PCR reaction (about 1.5 hours) then; Afterwards PCR product and the nitrocellulose filter that is marked with different probe (being respectively probe shown in sequence 1 to the sequence 4) are carried out water conservancy diversion hybridization (about 2 hours) through medical making nucleic acid molecular hybridization appearance; Result according to reaction can judge whether there is the Penicillium marneffei bacterium in the sample to be detected, and required time is 3.5 hours altogether.The result sees table 2.
The qualification result of Penicillium marneffei bacterium in table 2 mixed bacterium
| Bacterial classification in the associate strain sample | Detected result |
| Penicillium marneffei bacterium, Candida albicans and Candida parapsilosis | PM1, the positive A-P-SPEC1 of PM2, the A-P-SPEC2 positive |
Sequence table
< 110>Peking University First Hospital
< 120>application specific probe and the gene chip of evaluation Penicillium marneffei bacterium
<130>CGGNARY92638
<160>6
<210>1
<211>21
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>1
accggggcca?cccggtcgcc?g 21
<210>2
<211>22
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>2
tgtgaaccct?gatgaagatg?ga 22
<210>3
<211>19
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>3
tcctcgagcg?tatggggct 19
<210>4
<211>16
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>4
cggcttgtgt?gttggg 16
<210>5
<211>27
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>5
tccgtaggtg?aacctgcgga?aggatca 27
<210>6
<211>27
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>6
cgcttattga?tatgcttaag?ttcagcg 27
Claims (5)
1. identifying the application specific probe of Penicillium marneffei bacterium (Penicillium marneffei), is respectively that 4 dna fragmentations of sequence 1 to the sequence 4 of sequence table are formed by nucleotide sequence.
2. be used to identify the DNA chip of Penicillium marneffei bacterium (Penicillium marneffei), contain the said application specific probe of claim 1.
3. identify the device of Penicillium marneffei bacterium (Penicillium marneffei), comprise the said DNA chip of said application specific probe of claim 1 or claim 2.
4. device as claimed in claim 3 is characterized in that: said device comprises that also the primer that Nucleotide is formed shown in the sequence 6 of sequence 5 and sequence table of sequence table is right.
5. device as claimed in claim 4 is characterized in that: said primer has vitamin H to marked.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102372277A CN101705300B (en) | 2009-11-05 | 2009-11-05 | Special probe and gene chip used for identifying penicillium marneffei |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102372277A CN101705300B (en) | 2009-11-05 | 2009-11-05 | Special probe and gene chip used for identifying penicillium marneffei |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101705300A CN101705300A (en) | 2010-05-12 |
| CN101705300B true CN101705300B (en) | 2012-06-06 |
Family
ID=42375552
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009102372277A Expired - Fee Related CN101705300B (en) | 2009-11-05 | 2009-11-05 | Special probe and gene chip used for identifying penicillium marneffei |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101705300B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509870B (en) * | 2013-09-29 | 2015-04-08 | 广西医科大学 | One group of primers and probes for diagnosing penicilliosis marneffei and application thereof |
| CN104726568A (en) * | 2015-03-04 | 2015-06-24 | 何志义 | Penicillium marneffei LAMP (loop-mediated isothermal amplification) kit and application thereof |
| CN106676193B (en) * | 2017-03-06 | 2020-07-24 | 山西省农业科学院食用菌研究所 | Molecular marker, primer and probe for identifying penicillium |
| CN112626182A (en) * | 2021-02-01 | 2021-04-09 | 云南省传染病医院、云南省艾滋病关爱中心(云南省心理卫生中心) | Molecular identification method of Marneffei staphylium |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050260584A1 (en) * | 2001-09-26 | 2005-11-24 | Lindsley Mark D | Nucleic acids for the identification of fungi and methods for using the same |
| JP2005341955A (en) * | 2004-06-07 | 2005-12-15 | Chiba Univ | New polynucleotide, marker, probe and primer each for detecting penicillium marneffei as pathogen of deep mycosis using the same, method for detecting the pathogen, and kit for the method |
| CN101492743A (en) * | 2009-01-19 | 2009-07-29 | 中国人民解放军第三军医大学 | Pathogenic epiphyte detection gene chip |
-
2009
- 2009-11-05 CN CN2009102372277A patent/CN101705300B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050260584A1 (en) * | 2001-09-26 | 2005-11-24 | Lindsley Mark D | Nucleic acids for the identification of fungi and methods for using the same |
| JP2005341955A (en) * | 2004-06-07 | 2005-12-15 | Chiba Univ | New polynucleotide, marker, probe and primer each for detecting penicillium marneffei as pathogen of deep mycosis using the same, method for detecting the pathogen, and kit for the method |
| CN101492743A (en) * | 2009-01-19 | 2009-07-29 | 中国人民解放军第三军医大学 | Pathogenic epiphyte detection gene chip |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101705300A (en) | 2010-05-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lau et al. | Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens | |
| CN105154589B (en) | A kind of multi-fluorescence immunoassay method of quick differentiation PEDV, TGEV, PoRV | |
| Wahyuningsih et al. | Simple and rapid detection of Candida albicans DNA in serum by PCR for diagnosis of invasive candidiasis | |
| WO2012169099A1 (en) | Method for detecting fungi, reaction solution for pcr, and carrier for detecting fungi | |
| CN105112519A (en) | CRISPR-based Escherichia coli O157:H7 strain detection reagent box and detection method | |
| CN101638688B (en) | Special probe and gene chip for identifying pathogenic candida | |
| CN101492743A (en) | Pathogenic epiphyte detection gene chip | |
| CN101705300B (en) | Special probe and gene chip used for identifying penicillium marneffei | |
| CN101705301B (en) | Special probe and gene chip used for identifying pathogenic aspergillus | |
| CN106834506A (en) | A kind of LAMP primer group of quick detection bacterium polymyxins drug resistant gene mcr 1, kit and detection method | |
| CN102925562A (en) | Kit and method for detecting aminoglycoside drug-induced deafness-sensitive gene | |
| Brandt et al. | Laboratory aspects of medical mycology | |
| CN107164526A (en) | A kind of method for visualizing genechip detection Pathogen of Lung Infection | |
| CN104263833B (en) | Nucleic acid film bar and test kit for candidiasis strain identification and detection in Gene Mutation | |
| CN103194536A (en) | Nucleic acid chromatography detection kit of Candida albicans, detection method and application thereof | |
| CN101348829B (en) | Gene chip for detecting Mycotoruloides | |
| CN105112558B (en) | The triple real time fluorescent quantitative RT PCR detection kits of the type of foot and mouth disease virus O, A and Asia I | |
| CN101724693B (en) | Primer pair for detecting nested PCR mycoplasmas, detection kit and use method thereof | |
| US8178343B2 (en) | Gene cluster diagnosis apparatus | |
| WO2016203740A1 (en) | Method for examining microorganism, kit for examining microorganism, microarray for examining microorganism, carrier for detecting mold, method for detecting mold and kit for detecting mold | |
| CN105331718A (en) | Gene chip and kit for detecting pathogenic bacteria in cerebrospinal fluid of patient with fungal infection of central nervous system | |
| Li et al. | Rapid identification of Candida spp. frequently involved in invasive mycoses by using flow-through hybridization and Gene Chip (FHGC) technology | |
| CN102417931B (en) | Polymerase chain reaction (PCR) fluorescence detection kit and detection method for candida albicans | |
| CN103014164A (en) | Duplex fluorescent quantitation RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for shellfish Bonamia and Perkinsus | |
| CN106048051B (en) | A kind of candida krusei fluorescence PCR detection reagent kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120606 Termination date: 20211105 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |