CN101705300A - Special probe and gene chip used for identifying penicillium marneffei - Google Patents
Special probe and gene chip used for identifying penicillium marneffei Download PDFInfo
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- CN101705300A CN101705300A CN200910237227A CN200910237227A CN101705300A CN 101705300 A CN101705300 A CN 101705300A CN 200910237227 A CN200910237227 A CN 200910237227A CN 200910237227 A CN200910237227 A CN 200910237227A CN 101705300 A CN101705300 A CN 101705300A
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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 acquired immune deficiency syndrome (AIDS) (AIDS) patient, the morbidity of opportunistic fungal infection is also in increase at full speed, and wherein the aggressive penicilliosis that is 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 medicine of correct diagnosis, choose reasonable sensitivity and formulate treatment plan and have crucial meaning.
During technology that directly detects fungi from clinical samples that molecular biology is relevant and method are constantly developing, the most Huo Yue 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, bronchoalveolar lavage fluid and fester that can increase.But, still can not detect the Penicillium marneffei bacterium in the clinical samples at present fast, specifically.
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 nucleic acid target molecule in the detected sample thereby detect hybridization complex by colour developing then.The hybridizing method that this technology is more traditional has high specificity, characteristics fast, if this technology and high-throughput low density gene chip technology and the high round pcr of susceptibility can 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 that sequence 1 to 4 dna fragmentations and the nucleotide sequence of sequence 4 of sequence table is respectively 4 dna fragmentations of the sequence 1 of sequence table to the reverse complementary sequence of sequence 4.More than 8 dna fragmentations, all be according to the design of the conserved sequence of Penicillium marneffei bacterium (Penicilliummarneffei), each dna fragmentation wherein, 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; comprise above arbitrary described application specific probe or above arbitrary described DNA chip. described device can comprise that also the primer that Nucleotide is formed shown in the sequence 6 of the sequence 5 of sequence table and sequence table is right. for the ease of detecting the results of hybridization of DNA to be measured and described probe, described primer on can be marked with vitamin H.
More than arbitrary described application specific probe, above 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 described 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 described 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 the sequence 5 of sequence table 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: described 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 the sequence 5 of sequence table and sequence table obtains target dna to carrying out pcr amplification; (2) target dna is hybridized with sequence 1 (PM1) to the dna fragmentation shown in the sequence 4 (PM2) of sequence table of sequence table respectively, pass judgment on as follows according to results of hybridization: the results of hybridization as infructescence 1 and/or sequence 2 is positive, contains Penicillium marneffei bacterium (Penicillium marneffei) in the described sample; Results of hybridization as infructescence 3 and/or sequence 4 is positive, may contain the Penicillium marneffei bacterium in the described 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: described 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 the sequence 5 of sequence table and sequence table obtains target dna to carrying out pcr amplification; (2) target dna is hybridized with sequence 1 to the dna fragmentation shown in the sequence 4 of sequence table of sequence table respectively, pass judgment on as follows according to results of hybridization: the results of hybridization as infructescence 1 and/or sequence 2 is not positive, does not contain Penicillium marneffei bacterium (Penicillium marneffei) in the described sample; Results of hybridization as infructescence 3 (A-P-SPEC1) and/or sequence 4 (A-P-SPEC2) is not positive, does not contain the Penicillium marneffei bacterium in the described 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 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 if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.
Dna fragmentation used in following examples is as follows:
Specific probe at 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 (being marked with vitamin H) on the primer:
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 as follows:
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 extracts genomic dna according to standard method respectively, use above-mentioned primer that (sequence 5 of sequence table and sequence 6) carried out PCR reaction (about 1.5 hours) then, afterwards PCR product and the nitrocellulose filter that is marked with different probe (being respectively sequence 1 to probe shown in the sequence 4) are carried out the water conservancy diversion hybridization by medical making nucleic acid molecular hybridization instrument, result according to reaction judges bacterial classification in the detected sample, and required time is 3.5 hours.The results are shown in Table 1.
The qualification result of table 1 Penicillium marneffei bacterium
| Sample | Detected result |
| Penicillium marneffei bacterium (Penicillium marneffei) | The positive A-P-SPEC1 of PM1, PM2, the A-P-SPEC2 positive |
| Candida albicans (Candida albicans) | The negative A-P-SPEC1 of PA1, PM2, A-P-SPEC2 feminine gender |
| Candida parapsilosis (Candida parapsilosis) | The negative A-P-SPEC1 of PM1, 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 of sequence table and sequence 6) carried out PCR reaction (about 1.5 hours) then, afterwards PCR product and the nitrocellulose filter that is marked with different probe (being respectively sequence 1 to probe shown in the sequence 4) are carried out water conservancy diversion hybridization (about 2 hours) by medical making nucleic acid molecular hybridization instrument, result according to reaction can judge whether there is the Penicillium marneffei bacterium in the detected sample, and required time is 3.5 hours altogether.The results are shown in Table 2.
The qualification result of Penicillium marneffei bacterium in table 2 mixed bacterium
| Bacterial classification in the mixed strains sample | Detected result |
| Penicillium marneffei bacterium, Candida albicans and Candida parapsilosis | The positive A-P-SPEC1 of PM1, 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 (10)
1. identify the application specific probe of Penicillium marneffei bacterium (Penicillium marneffei), be made up of in following 8 dna fragmentations at least one: nucleotide sequence is respectively that sequence 1 to 4 dna fragmentations and the nucleotide sequence of sequence 4 of sequence table is respectively 4 dna fragmentations of the sequence 1 of sequence table to the reverse complementary sequence of sequence 4.
2. be used to identify the DNA chip of Penicillium marneffei bacterium (Penicillium marneffei), contain the described application specific probe of claim 1.
3. identify the device of Penicillium marneffei bacterium (Penicillium marneffei), comprise the described DNA chip of described application specific probe of claim 1 or claim 2.
4. device as claimed in claim 3 is characterized in that: described device comprises that also the primer that Nucleotide is formed shown in the sequence 6 of the sequence 5 of sequence table and sequence table is right.
5. device as claimed in claim 4 is characterized in that: described primer on be marked with vitamin H.
6. the application of arbitrary described device in identifying Penicillium marneffei bacterium (Penicillium marneffei) in the described application specific probe of claim 1, the described DNA chip of claim 2 or the claim 3 to 5.
7. method of identifying Penicillium marneffei bacterium (Penicillium marneffei), be with the ITS district of the rDNA of bacterial strain to be measured respectively with the described application specific probe hybridization of claim 1, 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).
8. method as claimed in claim 7 is characterized in that: the ITS district of the rDNA of described bacterial strain to be measured is that the total DNA 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 the sequence 5 of sequence table and sequence table to carrying out.
9. one kind is detected the method that whether contains Penicillium marneffei bacterium (Penicillium marneffei) in the sample, and it is characterized in that: described 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 the sequence 5 of sequence table and sequence table obtains target dna to carrying out pcr amplification;
(2) target dna is hybridized with sequence 1 to the dna fragmentation shown in the sequence 4 of sequence table of sequence table respectively, pass judgment on as follows according to results of hybridization: the results of hybridization as infructescence 1 and/or sequence 2 is positive, contains Penicillium marneffei bacterium (Penicillium marneffei) in the described sample; Results of hybridization as infructescence 3 and/or sequence 4 is positive, may contain Penicillium marneffei bacterium (Penicillium marneffei) in the described sample.
10. one kind is detected the method that whether does not contain Penicillium marneffei bacterium (Penicillium marneffei) in the sample, and it is characterized in that: described 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 the sequence 5 of sequence table and sequence table obtains target dna to carrying out pcr amplification;
(2) target dna is hybridized with sequence 1 to the dna fragmentation shown in the sequence 4 of sequence table of sequence table respectively, passes judgment on as follows according to results of hybridization:
Results of hybridization as infructescence 1 and/or sequence 2 is not positive, does not contain Penicillium marneffei bacterium (Penicillium marneffei) in the described sample; Results of hybridization as infructescence 3 and/or sequence 4 is not positive, does not contain Penicillium marneffei bacterium (Penicillium marneffei) in the described sample.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509870A (en) * | 2013-09-29 | 2014-01-15 | 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 |
| CN106676193A (en) * | 2017-03-06 | 2017-05-17 | 山西省农业科学院食用菌研究所 | Molecular marker for identifying penicillium, primer and probe |
| 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 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7427472B2 (en) * | 2001-09-26 | 2008-09-23 | United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Methods for the differentiation and identification of medically important endemic fungi |
| JP4576489B2 (en) * | 2004-06-07 | 2010-11-10 | 国立大学法人 千葉大学 | Novel polynucleotide, marker, probe, primer, detection method and kit for detection of Penicillium marnefei, a causative fungus of penicillium using the same |
| CN101492743B (en) * | 2009-01-19 | 2012-04-18 | 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
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509870A (en) * | 2013-09-29 | 2014-01-15 | 广西医科大学 | One group of primers and probes for diagnosing penicilliosis marneffei and application thereof |
| 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 |
| CN106676193A (en) * | 2017-03-06 | 2017-05-17 | 山西省农业科学院食用菌研究所 | Molecular marker for identifying penicillium, primer and probe |
| 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 |
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