CN111560453A - Gene chip for identifying seven fungal infection pathogens, kit and detection method thereof - Google Patents

Gene chip for identifying seven fungal infection pathogens, kit and detection method thereof Download PDF

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CN111560453A
CN111560453A CN202010246173.7A CN202010246173A CN111560453A CN 111560453 A CN111560453 A CN 111560453A CN 202010246173 A CN202010246173 A CN 202010246173A CN 111560453 A CN111560453 A CN 111560453A
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gene chip
candida
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郑晓斌
姜昊
陈海洋
冯宇亮
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Suzhou Ruimaiying Genetic Testing Technology Co ltd
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Abstract

The invention discloses a gene chip for identifying seven fungal infection pathogens, which comprises a solid phase carrier and a gene chip probe fixed on the solid phase carrier; the gene chip probe comprises an oligonucleotide probe of a sample to be detected; the oligonucleotide probe of the sample to be detected comprises a Candida albicans probe, a Candida glabrata probe, a Candida parapsilosis probe, a Candida tropicalis probe, a Candida krusei probe, an Aspergillus fumigatus probe and an Aspergillus flavus virus probe, and the probe sequences are shown as SEQ ID NO. 1-7 in sequence. The invention successfully constructs a gene chip for identifying seven human fungal infection pathogens; each screening probe adopted by the gene chip prepared by the invention can be specifically combined with a corresponding pathogen target gene, and the hybridization signal is strong and stable, so that the gene chip has a wide application prospect.

Description

Gene chip for identifying seven fungal infection pathogens, kit and detection method thereof
Technical Field
The invention relates to the technical field of gene chips, in particular to a gene chip for identifying seven fungal infection pathogens, a kit and a detection method thereof.
Background
The fungi are part of the normal flora of human body, and exist in small amount on the oral cavity, upper respiratory tract, intestinal tract, urinary tract and skin surface of human body. Invasive fungal Infection (IFD) refers to a disease in which a conditionally pathogenic fungus invades the body, grows and multiplies in tissues, organs or blood, and causes inflammatory reaction and tissue damage when the host immune function is low or defective. The incidence and mortality of IFD in patients with hematological disorders, malignancies, organ transplantation and long-term use of broad-spectrum antibiotics, glucocorticoids, immunosuppressants etc. is on the rise.
The main pathogenic fungi for invasive fungal infection include conditional pathogenic fungi such as Candida, Aspergillus and Cryptococcus, and endemic pathogenic fungi such as histoplasma bacteria, dermatitis blastomyces and Sphaerotheca sp. The kind of the infected fungi is different under different environments. The definitive diagnosis of fungal infections requires histopathological evidence that patients with hematological malignancies often suffer from severe granulocytopenia and thrombocytopenia following chemotherapy and after allogeneic bone marrow transplantation and often cannot tolerate tissue biopsies. The traditional fungus detection method has low detection rate and detection time lag, influences early diagnosis and treatment of patients and leads to high mortality. The detection rate of the sputum culture fungi is only 8-34%, the positive rate of the bronchial lavage fluid fungi culture is only 45-62%, and the detection time is 3-4 days.
The current clinical commonly used techniques for detecting fungi mainly comprise: (1) microscopic examination, culture and identification: such as six-limb silver staining, PAS staining, silver ammonia G-S staining, 10% KOH method and fungus culture method are the most common clinical identification means, but direct microscopic examination has poor specificity and insufficient sensitivity, and the fungus species cannot be identified. Although the fungus culture can identify the fungus species, the culture condition is harsh, the culture time is long, the false negative is high, the sensitivity is insufficient, and the clinical treatment cannot be assisted. (2) Immunological methods: the basic principle of the immunological method is to utilize the specific combination of antigen and antibody to reach the mutual specific exploration. The method is mainly used for detecting the antigen, the antibody, the metabolite and the cell components of the fungus. Fungi can be classified according to their antigenicity. However, the specificity of antibodies has limited the development of this technology. Immunohistochemistry, enzyme-linked immunosorbent assay, immunoblotting and agglutination reaction are all based on the detection of fungi in different sections by an immunological method, but the G test is only widely applied to clinical tests, detects (1,3) -13-D-glucan in the cell wall of the fungi, and can continuously release the substance after phagocytic cells of a human body phagocytose the fungi, so that the content of blood and body fluid is increased (superficial fungal infection is not similar). It is suitable for early diagnosis of all deep fungal infections except cryptococcus and zygomycota, especially candida and fungal but not defined species. Clinical applications find many false positives possible, for example: the use of cellulose membranes for hemodialysis specimens or patients exposed to gauze or other dextran-containing material, intravenous infusions of immunoglobulins, albumins, clotting factors or blood products, streptococcinemia or contamination of the operator handling the specimens. In addition, the use of polysaccharide anticancer drugs, mucosal damage caused by radiotherapy and chemotherapy, which may cause glucan in food or implanted candida may enter blood through gastrointestinal tract, which may also cause false positive. (3) Molecular biology techniques: the method comprises in-situ hybridization, polymerase chain reaction, content determination of G + C mol% in DNA, Random Amplified Polymorphic DNA (RAPD) analysis, restriction endonuclease polymorphism analysis (RFLP) and the like, but the above techniques are time-consuming and labor-consuming, do not have the characteristic of high throughput, and cannot simultaneously and rapidly detect infected fungi by using a small amount of samples, so the method is mostly limited to laboratory research and cannot be really applied to clinic.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a gene chip for identifying seven fungal infection pathogens.
The invention also aims to provide a fungal infection pathogen detection and identification kit.
The invention further aims to provide a detection method of the kit for identifying seven fungal infection pathogens.
The above object of the present invention is achieved by the following technical solutions:
a gene chip for identifying seven fungal infection pathogens comprises a solid phase carrier and a gene chip probe fixed on the solid phase carrier; the gene chip probe comprises an oligonucleotide probe of a sample to be detected; the oligonucleotide probe of the sample to be detected comprises a Candida albicans probe, a Candida glabrata probe, a Candida parapsilosis probe, a Candida tropicalis probe, a Candida krusei probe, an Aspergillus fumigatus probe and an Aspergillus flavus virus probe, and the probe sequences are shown as SEQ ID NO. 1-7 in sequence.
Preferably, the gene chip probe also comprises a quality control oligonucleotide probe, which comprises a sample application position quality control probe and a hybridization positive quality control probe; the sequence of the sample application position quality control probe is shown as SEQ ID NO. 8; the hybridization positive quality control probe sequence is shown as SEQ ID NO. 9. Wherein, the quality control probe at the sample application position is a probe fixed positive control, the sample application process of the chip is monitored, and the chip is positive before and after hybridization; the hybridization positive quality control probe is a hybridization positive control, monitors the hybridization process of the chip, and detects that any one of the seven fungi is positive. The quality control also comprises a hybridization negative control which is a buffer solution without a probe and is used as the hybridization negative control, and any sample to be detected is negative.
Preferably, the 5' ends of the gene chip probes are connected with an amino group through 15 thymine nucleotides; that is, poly (dT) with a length of 15 is attached to the 5 'end of the probe as a spacer, and then an amino group is labeled to the 5' end.
Preferably, the gene chip probes are distributed and fixed on a solid phase carrier in a microarray mode; the solid phase carrier is a material which is conventional in the field, such as a silicon dioxide glass slide.
Preferably, the gene chip comprises a microarray of 8 rows by 7 columns; the distribution of the probes on the microarray is as follows from top to bottom: printing position quality control partition P1: 1 line × 8 dots; hybridization positive quality control partition P2: 1 line × 4 dots; hybridization negative quality control partition N: 1 line × 4 dots; partition 1 of candida albicans probe: 1 line × 4 dots; partition 2 of candida glabrata probe: 1 line × 4 dots; partition 3 of candida parapsilosis probe: 1 line × 4 dots; partition 4 of candida tropicalis probe: 1 line × 4 dots; partition 5 of the candida krusei probe: 1 line × 4 dots; partition 6 of the aspergillus fumigatus probe: 1 line × 4 dots; partition 7 of aspergillus flavus probe: 1 line × 4 dots; hybridization negative quality control partition N: 1 line × 4 dots; printing position quality control partition P1: 1 line x 8 dots.
Preferably, the gene core comprises at least one of the above-mentioned micro-needle arrays, and each micro-array can detect a sample.
The invention also provides a detection method of the gene chip in detecting seven fungal infection pathogens, which comprises the following steps:
s1, preparing a sample to be detected: extracting whole genome DNA/RNA of a sample to be detected, and preparing DNA/cDNA of the sample to be detected;
s2, PCR amplification: taking the DNA/cDNA of the sample to be detected in the step S1 as a template, and carrying out PCR amplification reaction by using a fungal gene amplification primer pair shown in SEQ ID NO. 10-11;
s3, hybridization: hybridizing a hybridization positive control, the PCR product of step S2 and the gene chip:
s4, interpretation of results: and scanning and analyzing the hybridized gene chip to judge the result.
Specifically, step S1 is to extract the whole genome DNA/RNA of the sample to be tested according to the instruction of commercial DNA/RNA extraction kit, or to extract according to the conventional method in the prior art.
Preferably, the PCR amplification reaction system in step S2 includes 25 μ L of PCR reaction solution: 12.5 mu L of rTaq enzyme, 2 mu L of primer Mix, 5 mu L of DNA/cDNA of a sample to be detected and 5.5 mu L of nuclease-free sterilized water; wherein the primer Mix contains 1 muL of upstream primer of 20 mumol/L fungus gene amplification (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Aspergillus fumigatus and Aspergillus flavus) with the sequence of SEQ ID NO. 10, and 1 muL of downstream primer of 20 mumol/L fungus gene amplification (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Aspergillus fumigatus and Aspergillus flavus) with the sequence of SEQ ID NO. 11.
Preferably, the PCR amplification reaction procedure in step S2 is: 5min at 94 ℃; circulating for 12 times at 94 ℃ for 30sec, 58 ℃ for 30sec and 72 ℃ for 25 sec; 30 cycles at 94 ℃ for 30sec, 50 ℃ for 30sec, and 72 ℃ for 25 sec; 5min at 72 ℃.
Preferably, the hybridization in step S3 is to mix 8 μ L of PCR amplification product and 0.1 μ L of hybridization positive control, denature for 8min at 95 ℃, stand for 5min on ice, and then take 15 μ L of hybridization solution to perform hybridization.
The invention also provides application of the gene chip in preparation of a fungal infection pathogen detection and identification kit.
The invention also provides a fungal infection pathogen detection and identification kit, and the gene chip.
Preferably, the kit further comprises a PCR reaction mixture, Taq polymerase, PCR positive control, PCR negative control, ddH20. Chip cover plate, hybridization solution, hybridization positive control and hybridization box; the PCR reaction mixed solution comprises a primer Mix containing 20 mu mol/L fungal gene amplification upstream primer with a sequence shown as SEQ ID NO. 10 and 20 mu mol/L fungal gene amplification downstream primer with a sequence shown as SEQ ID NO. 11.
Preferably, the hybridization solution comprises 20 × SSC 500. mu.L, 10% SDS 10. mu.L and DEPC H per 1000. mu.L2O 490μL。
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention successfully constructs a gene chip for identifying infection pathogeny of seven human fungi (candida albicans, candida glabrata, candida parapsilosis, candida tropicalis, candida krusei, aspergillus fumigatus and aspergillus flavus): each screening probe adopted by the gene chip prepared by the invention can be specifically combined with a corresponding pathogen target gene, and the hybridization signal is strong and stable.
(2) The gene detection chip prepared by the invention has good methodological characteristics: the invention establishes a gene chip detection method with good specificity, high sensitivity, strong stability and time and labor saving.
(3) The construction of the detection gene chip provides a rapid and efficient means for detection and differential diagnosis of fungal infection diseases and provides technical support for pathogen detection of fungal infection patients.
Drawings
FIG. 1 is a schematic diagram showing the distribution of probes of the gene chip of the present invention.
FIG. 2 shows the result of detecting Candida albicans hybridization using the gene chip of the present invention; the result shows that the product of Candida albicans genome DNA after PCR amplification can be combined with a detection probe, and the No. 1 probe is bright except that the quality control probe meets the requirement, which indicates that the gene chip can be used for detecting Candida albicans.
FIG. 3 shows the result of hybridization for detecting Candida glabrata using the gene chip of the present invention; the result shows that the product of Candida glabrata genome DNA after PCR amplification can be combined with a detection probe, and the No. 2 probe is bright except that the quality control probe meets the requirement, which indicates that the gene chip can be used for detecting Candida glabrata.
FIG. 4 shows the result of detecting Candida parapsilosis hybridization using the gene chip of the present invention; the result shows that the product of Candida parapsilosis genome DNA after PCR amplification can be combined with a detection probe, and the No. 3 probe is bright except that the quality control probe meets the requirement, which indicates that the gene chip can be used for detecting Candida parapsilosis.
FIG. 5 shows the result of detecting Candida tropicalis hybridization using the gene chip of the present invention; the result shows that the product of Candida tropicalis genome DNA after PCR amplification can be combined with a detection probe, and the No. 4 probe is bright except that the quality control probe meets the requirement, which indicates that the gene chip can be used for detecting Candida tropicalis.
FIG. 6 shows the result of detecting Candida krusei hybridization using the gene chip of the present invention; the result shows that the product of PCR amplification of Candida krusei genome DNA can be combined with a detection probe, and the No. 5 probe is bright except that the quality control probe meets the requirement, which indicates that the gene chip can be used for detecting Candida krusei.
FIG. 7 shows the result of hybridization of Aspergillus fumigatus using the gene chip of the present invention; the result shows that the product of the PCR amplification of the aspergillus fumigatus genome DNA can be combined with a detection probe, and the No. 6 probe is bright except that the quality control probe meets the requirement, which indicates that the gene chip can be used for detecting the aspergillus fumigatus.
FIG. 8 shows the hybridization results of Aspergillus flavus detection using the gene chip of the present invention; the result shows that the product of the Aspergillus flavus genome DNA after PCR amplification can be combined with a detection probe, and the No. 7 probe is bright except that the quality control probe meets the requirement, which indicates that the gene chip can be used for detecting the Aspergillus flavus.
FIG. 9 shows the hybridization results of seven pathogenic bacteria; the result shows that after the PCR amplification products of the seven pathogenic bacteria are hybridized, the No. 1-7 probes are bright except that the quality control probes meet the requirements, and the gene chip can be used for detecting the seven pathogenic fungi.
In the figure: p1-sample application position quality control partition; p2-hybridization positive quality control subarea; n-hybridization negative quality control subareas; 1-partition of Candida albicans probe; 2-partition of candida glabrata probes; 3-partitioning of Candida parapsilosis probes; 4-partitioning of candida tropicalis probes; 5-partitioning of Candida krusei probes; 6-partitioning of the Aspergillus fumigatus probe; 7-partitioning of the Aspergillus flavus probe.
Description of hybridization: p1: the positive control is fixed by the probe, the chip sample application process is monitored, and the chip is positive before and after hybridization.
P2: and (3) hybridization positive control, monitoring the chip hybridization process, and detecting that any sample is positive.
N: hybridization negative control, any sample detected should be negative.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
EXAMPLE 1 extraction of DNA from test sample
The extraction can be performed according to the routine method in the field, and the sample can be fresh tissue fluid, blood, urine, cerebrospinal fluid, secretion (sputum, pleural fluid, ascites, alveolar lavage fluid, etc.). In this example, using the Qiagen miniprep DNA extraction kit, fresh tissue is used as an example:
(1) putting fresh tissues into a sterile mortar, pouring liquid nitrogen for freezing, then grinding, and transferring the ground tissues into a 1.5mL EP tube;
(2) adding 1ml PBS buffer, washing, centrifuging at 3000rpm for 5 minutes, and discarding the supernatant;
(3) adding 200 μ L of 50mM NaOH solution, incubating at 95 deg.C for 10 min, centrifuging at 5000rpm for 10 min, and discarding the supernatant;
(4) adding 500 mu L of Lyticase muramidase solution, incubating for 30 minutes at 37 ℃, centrifuging for 10 minutes at 14000rpm, and removing the supernatant;
(5) adding 180. mu.L ATL and 20. mu.L proteinase K quickly, and incubating at 55 deg.C for 15 min;
(6) adding 100 μ L AL (containing 1 μ g carrier RNA), shaking for 15 seconds, mixing well, and incubating at 70 deg.C for 10 min;
(7) adding 50 mu L of absolute ethyl alcohol, shaking for 15 seconds, uniformly mixing, and standing at room temperature for 5 minutes;
(8) moving the uniformly mixed product to a column to avoid wetting the tube wall, centrifuging at 8000rpm for 1 minute, putting the column into a new 2mL collecting tube, and if the product does not completely pass through the membrane, centrifuging at a higher rotating speed again until the product completely passes through the membrane;
(9) add 500. mu.L AW1 to the column, avoid wetting the tube wall, centrifuge at 8000rpm for 1 minute, place the column into a new 2mL collection tube;
(10) add 500. mu.L AW2 to the column, avoid wetting the tube wall, centrifuge at 8000rpm for 1 minute, place the column into a new 2mL collection tube;
(11) centrifuge at 14000rpm for 3 minutes to completely dry the membrane to ensure no ethanol;
(12) placing the column in a 1.5mL centrifuge tube, adding 20-100 mu LAE distilled water to the center of the membrane, and placing for 1 minute at room temperature;
(13) the column was placed on a collection tube and centrifuged at 14000rpm for 1 minute to preserve the liquid in the tube for use.
EXAMPLE 2 PCR amplification of fungal rDNA-ITS sequences in samples to be tested
The PCR reaction system comprises: 12.5 mu L of rTaq enzyme, 2 mu L of primer Mix, 5 mu L of DNA/cDNA of a sample to be detected and 5.5 mu L of nuclease-free sterilized water;
wherein the primer Mix contains: 20 mu mol/L of the universal primer upstream primer (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Aspergillus fumigatus and Aspergillus flavus) with the sequence of SEQ ID NO. 10, and 1 mu L of the universal primer downstream primer (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Aspergillus fumigatus and Aspergillus flavus) with the sequence of SEQ ID NO. 11.
SEQ ID NO:10：
5’-TCCGTAGGTGAACCTGCGG-3’；
SEQ ID NO:11：
5’-TCCTCCGCTTATTGATATGC-3’；
Amplification was performed as follows: 5min at 94 ℃; circulating for 12 times at 94 ℃ for 30sec, 58 ℃ for 30sec and 72 ℃ for 25 sec; 30 cycles at 94 ℃ for 30sec, 50 ℃ for 30sec, and 72 ℃ for 25 sec; 5min at 72 ℃; is used for amplifying the fungal rDNA-ITS sequence of a sample to be detected.
Example 3 fungal specific Probe design
The invention respectively carries out sequence analysis on genome DNA of Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Aspergillus fumigatus and Aspergillus flavus, designs 7 oligonucleotide probes which can be specifically combined with a PCR product (rDNA-ITS) of a corresponding pathogen, when the PCR carries out genome amplification, the 5' end of an upstream universal primer is marked by Cy3 fluorescent pigment, and the probes can be hybridized with the product at 42 ℃. And judging the hybridization result according to the position and the intensity of the fluorescent signal so as to identify the infection of candida albicans, candida glabrata, candida parapsilosis, candida tropicalis, candida krusei, aspergillus fumigatus and aspergillus flavus. The universal PCR primers SEQ ID NO 10 and SEQ ID NO 11 were designed using PrimerPremier5.0 software based on the entire gene sequences of Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Aspergillus fumigatus and Aspergillus flavus published on the Genbank website. Meanwhile, gene sequences are cloned, sequencing, comparison analysis and primer design are carried out, and 7 specific oligonucleotide probes, namely SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7, are finally obtained through a large number of screening tests. Through the two primers and the 7 specific probes, the 7 pathogens can be identified and distinguished purposely and sensitively and rapidly.
Meanwhile, quality control sequences SEQ ID NO 8 and SEQ ID NO 9 are synthesized according to the gene sequences of the 7 fungi and can be hybridized and combined with PCR amplification products of all the 7 fungi.
The 5' end of each probe is linked with an amino group through 15 thymine nucleotide chains, and the specific nucleotide sequence is as follows:
SEQ ID NO:1：
5’-NH2-T15-CAGCGGTTTGAGGGAGAAAC-3’；
SEQ ID NO:2：
5’-NH2-T15-CCCTCCCTAGATCAACACCG-3’；
SEQ ID NO:3：
5’-NH2-T15-GGCCTTCTATATGGGGCCTGCCA-3’；
SEQ ID NO:4：
5’-NH2-T15-TTATAACCTCTGGCGGTGGG-3’；
SEQ ID NO:5：
5’-NH2-T15-GCCTTCCACACAGACTCCAA-3’；
SEQ ID NO:6：
5’-NH2-T15-CAGGTGACAAAGCCCCATAC-3’；
SEQ ID NO:7：
5’-NH2-T15-GGAGACACCACGAACTCTGT-3’；
SEQ ID NO:8：
NH2-T15-GCTGCCTCGG CAAGGAGT-TAMRA；
SEQ ID NO:9：
NH2-T15-GAATACCAGA GGGCGCAATG。
EXAMPLE 4 preparation of Gene chip
Referring to fig. 1, a gene chip for detecting seven important fungal infection pathogens adopts a gene chip micro spotting technology to fix a sample probe to be detected and various quality control probes on a chemically modified substrate to form a microarray with 8 rows by 7 columns. The distribution of the probes on the microarray is as follows from top to bottom:
printing position quality control partition P1: 1 line × 8 dots;
hybridization positive quality control partition P2: 1 line × 4 dots;
hybridization negative quality control partition N: 1 line × 4 dots;
partition 1 of candida albicans probe: 1 line × 4 dots;
partition 2 of candida glabrata probe: 1 line × 4 dots;
partition 3 of candida parapsilosis probe: 1 line × 4 dots;
partition 4 of candida tropicalis probe: 1 line × 4 dots;
partition 5 of the candida krusei probe: 1 line × 4 dots;
partition 6 of the aspergillus fumigatus probe: 1 line × 4 dots;
partition 7 of aspergillus flavus probe: 1 line × 4 dots;
hybridization negative quality control partition N: 1 line × 4 dots;
printing position quality control partition P1: 1 line x 8 dots.
Each chip has at least one microarray, each microarray being capable of detecting a sample.
The hybridization negative control zone N contains spotting buffer, which is a commercially available product, such as that produced by Boo biological Limited. The other partitions comprise corresponding detection probes and quality control probes.
EXAMPLE 5 Gene chip reagent preparation
Chip washing liquid
According to the needs and the actual situation, the washing liquid I and the washing liquid II are prepared according to the following proportion.
Washing solution I: the final concentration of SSC was 2X and the final concentration of SDS was 0.2%. For example, 600mL wash I: 528mL of distilled water +60mL of 20X SSC +12mL of 10% SDS, or formulated in proportions as required.
Washing solution II: the final concentration of SSC was 0.2X and the final concentration of SDS was 0.2%. For example, 600mL wash I: 594mL of distilled water +6mL of 20 XSSC, or formulated in proportions as required.
EXAMPLE 6 sample detection and interpretation
1. And (3) film washing: melting the hybridization buffer solution at 42 ℃, preparing the hybridization solution according to the following surface system, denaturing the mixed solution of the hybridization system at 95 ℃ for 8 minutes, and carrying out ice bath for 5 minutes for later use. Preparing 16 mu L of hybridization solution: hybridization buffer 7.9. mu. L, PCR amplification product 8. mu.L, hybridization positive control 0.1. mu.L.
The chip hybridization box was opened, and the hybridization box was placed flat on a table, and about 200. mu.L of sterile water was added to the bottom recess of the hybridization box. Placing the chip with the right side facing upwards between two positioning pins in the hybridization box, placing a chip cover plate, paying attention to that the side with the boss faces towards the chip, contacting the chip at the upper end, and slowly covering the chip; then, a pipette is used to slowly inject 15 μ L of denatured hybridization solution through the cover plate well, and the hybridization solution will form a liquid film between the boss under the cover plate and the chip surface by virtue of the liquid surface tension. Care was taken not to shake the cover or chip to avoid damaging the liquid film. And (5) tightly covering the hybridization box cover. And (3) placing the mixture into a constant-temperature water bath at 42 ℃, standing and hybridizing for 2-3 hours.
After hybridization, the chip is taken out and placed in washing liquor I preheated at 42 ℃, shaking and washing are carried out for 5 minutes at 42 ℃, then washing liquor II preheated at 42 ℃ is used, shaking and washing are carried out for 5 minutes at 42 ℃ (washing liquor II can be washed twice in winter, and is washed for two minutes each time), finally washing is carried out once with clear water preheated at 42 ℃, the washed chip is centrifuged at 2000rpm for 2 minutes to remove liquid on the surface of the chip, the chip can be stored in a dark place, and scanning is effective within 4 hours.
2. Scanning and result interpretation
The cleaned chip was subjected to scanning analysis using a microarray scanner with a LuxScan 10K-A microarray scanner, the background of negative control was removed, and the result was judged by comparing with the chip hybridization instructions.
FIGS. 2 to 9 are graphs showing the results of detection according to the embodiment of the present invention, which are the results of hybridization of PCR amplification products of Candida albicans genomic DNA; the hybridization result of the PCR amplification product of Candida glabrata genome DNA; the hybridization result of the PCR amplification product of the Candida parapsilosis genome DNA; hybridizing a PCR amplification product of Candida tropicalis genome DNA; hybridizing a PCR amplification product of Candida krusei genome DNA; hybridizing the PCR amplification product of the aspergillus fumigatus genome DNA; and (3) hybridizing the Aspergillus flavus genome DNA through a PCR amplification product. The gene chip can be used for detecting candida albicans, candida glabrata, candida parapsilosis, candida tropicalis, candida krusei, aspergillus fumigatus and aspergillus flavus respectively or simultaneously.
Sequence listing
<110> Sci Rui Mei Ying Gene detection technology Co., Ltd, Suzhou
<120> gene chip for identifying seven fungal infection pathogens, kit and detection method thereof
<141>2020-03-31
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Claims (10)

1. A gene chip for identifying seven fungal infection pathogens is characterized by comprising a solid phase carrier and a gene chip probe fixed on the solid phase carrier; the gene chip probe comprises an oligonucleotide probe of a sample to be detected; the oligonucleotide probe of the sample to be detected comprises a Candida albicans probe, a Candida glabrata probe, a Candida parapsilosis probe, a Candida tropicalis probe, a Candida krusei probe, an Aspergillus fumigatus probe and an Aspergillus flavus virus probe, and the probe sequences are shown as SEQ ID NO. 1-7 in sequence.
2. The gene chip of claim 1, wherein the gene chip probes further comprise quality control oligonucleotide probes comprising spotting position quality control probes, hybridization positive quality control probes; the sequence of the quality control probe at the sample application position is shown as SEQID NO. 8; the hybridization positive quality control probe sequence is shown as SEQ ID NO. 9.
3. The gene chip of claim 1 or 2, wherein the 5' ends of the probes of the gene chip are connected with an amino group through 15 thymine nucleotides.
4. The gene chip of claim 1 or 2, wherein the gene chip probes are distributed and immobilized on a solid phase carrier in a microarray manner.
5. The gene chip of claim 4, wherein the gene chip comprises a microarray of 8 rows by 7 columns; the distribution of the probes on the microarray is as follows from top to bottom: printing position quality control partition P1: 1 line × 8 dots; hybridization positive quality control partition P2: 1 line × 4 dots; hybridization negative quality control partition N: 1 line × 4 dots; partition 1 of candida albicans probe: 1 line × 4 dots; partition 2 of candida glabrata probe: 1 line × 4 dots; partition 3 of candida parapsilosis probe: 1 line × 4 dots; partition 4 of candida tropicalis probe: 1 line × 4 dots; partition 5 of the candida krusei probe: 1 line × 4 dots; partition 6 of the aspergillus fumigatus probe: 1 line × 4 dots; partition 7 of aspergillus flavus probe: 1 line × 4 dots; hybridization negative quality control partition N: 1 line × 4 dots; printing position quality control partition P1: 1 line x 8 dots.
6. The method for detecting seven fungal infection pathogens by using the gene chip of any one of claims 1 to 5, which is characterized by comprising the following steps:
s1, preparing a sample to be detected: extracting whole genome DNA/RNA of a sample to be detected, and preparing DNA/cDNA of the sample to be detected;
s2, PCR amplification: taking the DNA/cDNA of the sample to be detected in the step S1 as a template, and carrying out PCR amplification reaction by using a fungal gene amplification primer pair shown in SEQ ID NO. 10-11;
s3, hybridization: hybridizing a hybridization positive control, the PCR product of step S2 and the gene chip:
s4, interpretation of results: and scanning and analyzing the hybridized gene chip to judge the result.
7. Use of the gene chip of claims 1-5 in the preparation of a fungal infection pathogen detection and identification kit.
8. A kit for detecting and identifying a fungal infection pathogen, comprising the gene chip according to any one of claims 1 to 5.
9. The kit of claim 8, further comprising a PCR reaction mixture, Taq polymerase, PCR positive control, PCR negative control, ddH20. Chip cover plate, hybridization solution, hybridization positive control and hybridization box; the PCR reaction mixed solution comprises a primer Mix containing 20 mu mol/L fungal gene amplification upstream primer with a sequence shown as SEQ ID NO. 10 and 20 mu mol/L fungal gene amplification downstream primer with a sequence shown as SEQ ID NO. 11.
10. The kit of claim 9, wherein the hybridization solution comprises 20 × SSC 500. mu.L, 10% SDS 10. mu.L and DEPC H per 1000. mu.L2O 490μL。
CN202010246173.7A 2020-03-31 2020-03-31 Gene chip for identifying seven fungal infection pathogens, kit and detection method thereof Pending CN111560453A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1696311A (en) * 2005-04-22 2005-11-16 中国人民解放军军事医学科学院卫生学环境医学研究所 Biochip for detecting pathogenesis fungus
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
CN105063237A (en) * 2015-08-07 2015-11-18 重庆出入境检验检疫局检验检疫技术中心 Gene chip for identification of six swine disease pathogens and detection method thereof
CN105063760A (en) * 2015-08-07 2015-11-18 重庆出入境检验检疫局检验检疫技术中心 Gene chip for identification of seven swine disease pathogens and detection method thereof
CN110863037A (en) * 2019-11-15 2020-03-06 北京贝尔生物工程股份有限公司 Microarray chip capable of detecting various fungal genome DNAs (deoxyribonucleic acids) and preparation method thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1696311A (en) * 2005-04-22 2005-11-16 中国人民解放军军事医学科学院卫生学环境医学研究所 Biochip for detecting pathogenesis fungus
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
CN105063237A (en) * 2015-08-07 2015-11-18 重庆出入境检验检疫局检验检疫技术中心 Gene chip for identification of six swine disease pathogens and detection method thereof
CN105063760A (en) * 2015-08-07 2015-11-18 重庆出入境检验检疫局检验检疫技术中心 Gene chip for identification of seven swine disease pathogens and detection method thereof
CN110863037A (en) * 2019-11-15 2020-03-06 北京贝尔生物工程股份有限公司 Microarray chip capable of detecting various fungal genome DNAs (deoxyribonucleic acids) and preparation method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
顾大勇等: "以纳米金为报告系统的病原体快速检测基因芯片的研制", 《中华医院感染学杂志》 *

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Application publication date: 20200821