Nucleic acid detection kit and detection method for multiple candida

Abstract

The invention relates to a multiple candida nucleic acid detection kit and a detection method, which can simultaneously realize combined qualitative detection of five pathogenic fungi, namely candida albicans, candida krusei, candida glabrata, candida tropicalis and candida parapsilosis. Specific primers and specific probes of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis are utilized, and polymerase chain reaction and fluorescent probe technologies are combined. Amplification detection is respectively carried out on high-conservative-specificity sequences of candida albicans, candida krusei, candida glabrata, candida tropicalis and candida parapsilosis, and qualitative detection is carried out on the five bacteria. The method has the advantages of high detection specificity, high sensitivity, high accuracy and high detection efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a multiple Candida nucleic acid detection kit and a detection method. Background Art

[0002] In recent years, more and more cases of Candida infection have occurred, especially in patients with weakened immune function, such as cancer patients, HIV-infected patients, and organ transplant recipients. Candida infection not only affects the patient's quality of life, but may also cause serious complications and even endanger life. According to statistics from the World Health Organization (WHO), there are about millions of cases of Candida infection worldwide each year, of which about 40% occur in patients with weakened immune function. These patients are more susceptible to Candida invasion due to their weakened immune system, and are more difficult to cure after infection, and the mortality rate is relatively high.

[0003] Among the Candida genus, Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, and Candida parapsilosis are the most common pathogenic species in clinical practice. Different species of Candida have different sensitivities to antifungal drugs. For example, Candida krusei is naturally resistant to fluconazole, while Candida glabrata often exhibits multidrug resistance. Therefore, when choosing antifungal treatment options, clinicians must make the most appropriate decision based on the specific Candida species. Therefore, accurate and rapid identification and typing of these Candida species are of great significance for clinical diagnosis and treatment.

[0004] Existing methods for detecting Candida have many shortcomings in clinical applications. Traditional culture methods take several days to obtain results, and have limited sensitivity and specificity, which are prone to missed detections and false detections. Although microscopic examination can observe Candida in a short period of time, it cannot accurately type and requires professional technicians to operate. Nonspecific staining may occur in the immunofluorescence method, resulting in background fluorescence interference. Nonspecific staining increases the difficulty of interpreting the results and affects the sensitivity and specificity of the test. In addition, the immunofluorescence method has high requirements for the storage and handling of samples, and the samples need to maintain good morphology and antigenicity. Improper storage or improper handling of samples may lead to antigen degradation or loss, affecting the accuracy of the test results. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a multiple Candida nucleic acid detection kit and a detection method.

[0006] The technical solution adopted by the present invention is: a primer probe set for detecting multiple Candida species, comprising a specific forward primer, a specific reverse primer and a specific probe for respectively detecting at least two species of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis; Among them, the specific forward primer, specific reverse primer and specific probe sequences for detecting Candida albicans are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 respectively; The specific forward primer, specific reverse primer and specific probe sequences for detecting Candida krusei are shown in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 respectively; The specific forward primer, specific reverse primer and specific probe sequences for detecting Candida glabrata are shown in SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9 respectively; The specific forward primer, specific reverse primer and specific probe sequences for detecting Candida tropicalis are shown in SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12 respectively; The specific forward primer, specific reverse primer and specific probe sequences for detecting Candida parapsilosis are shown in SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No. 15 respectively.

[0007] Preferably, the Candida albicans-specific probe and the Candida krusei-specific probe contain a first fluorescent reporter gene; the Candida glabrata-specific probe contains a second fluorescent reporter gene; and the Candida tropicalis-specific probe and the Candida parapsilosis-specific probe contain a third fluorescent reporter gene.

[0008] Preferably, the first fluorescent reporter gene, the second fluorescent reporter gene and the third fluorescent reporter gene are FAM, VIC and CY5 respectively; and the Candida albicans-specific probe, the Candida krusei-specific probe, the Candida glabrata-specific probe, the Candida tropicalis-specific probe and the Candida parapsilosis-specific probe all include MGB.

[0009] Preferably, the method comprises specific forward primers, specific reverse primers and specific probes for detecting Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis, respectively.

[0010] A multiple Candida nucleic acid detection kit comprises a primer probe set for detecting multiple Candida.

[0011] Preferably, it also includes a positive quality control product, a negative quality control product and a detection reaction solution, wherein the detection reaction solution includes a primer probe set; Preferably, the positive quality control product includes artificial simulated alveolar lavage fluid, a solution containing the genome of Candida albicans, a solution containing the genome of Candida krusei, a solution containing the genome of Candida glabrata, a solution containing the genome of Candida tropicalis, and a solution containing the genome of Candida parapsilosis; the negative quality control product is sterile enzyme-free deionized water.

[0012] Preferably, it also includes a specific forward primer, a specific reverse primer and a specific probe for detecting the internal reference gene RNase P, and the sequences are shown in SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18 respectively.

[0013] A method for detecting multiple types of Candida species, using a primer probe set for detecting multiple types of Candida species, or using a multiple types of Candida nucleic acid detection kit for detection.

[0014] Preferably, the Candida species in the sample are qualitatively identified based on whether the characteristic peak of the fluorescent reporter gene appears and the position of the characteristic peak.

[0015] Preferably, the Candida albicans-specific probe and the Candida krusei-specific probe contain a first fluorescent reporter gene; in the first fluorescent reporter gene color development channel, if the Tm value of the characteristic peak is between 71.5 and 74.5°C, it is determined to be Candida albicans positive; if the Tm value is between 78.3 and 80.7°C, it is determined to be Candida krusei positive; if there is no characteristic peak or the Tm value is not between 71.5 and 74.5°C and 78.3 and 80.7°C, it is Candida albicans negative and Candida krusei negative; The Candida glabrata specific probe contains a second fluorescent reporter gene; in the second fluorescent reporter gene color development channel, if the Tm value of the characteristic peak is between 72.5 and 74.7°C, it is determined to be Candida glabrata positive; if there is no characteristic peak or the Tm value is not between 72.5 and 74.7°C, it is determined to be Candida glabrata negative; The Candida tropicalis-specific probe and the Candida parapsilosis-specific probe contain a third fluorescent reporter gene. In the second fluorescent reporter gene color development channel, if the Tm value of the characteristic peak is between 70.2 and 72.1°C, it is determined to be positive for Candida tropicalis; if the Tm value is between 76.5 and 78.7°C, it is determined to be positive for Candida parapsilosis; if there is no characteristic peak or the Tm value is not between 70.2 and 72.1°C and 76.5 and 78.7°C, it is negative for Candida tropicalis and negative for Candida parapsilosis.

[0016] The advantages and positive effects of the present invention are as follows: providing a set of primer sets and a kit capable of simultaneously detecting a variety of Candida species, designing primers and probes specifically targeting Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis, adding a fluorescent reporter gene to the probe, and combining the fluorescence color development and peak position to qualitatively classify the types of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis; the method has high detection specificity, high sensitivity, high accuracy and high detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The specific result diagram of fluorescent PCR detection (FAM channel); 1 is Candida albicans, 2 is Candida krusei; Figure 2 The specific result graph of fluorescence PCR detection (VIC channel) is shown; 3 is Candida glabrata; Figure 3 The specific result graph of the fluorescence PCR test (CY5 channel) is shown; 4 is Candida tropicalis, 5 is Candida parapsilosis; Figure 4 Specific experimental internal reference detection channel detection result diagram (ROX channel); Figure 5 Sensitivity results of fluorescent PCR detection of Candida albicans (FAM channel); 1. Sample concentration is 1000 spores / mL, 2. Sample concentration is 500 spores / mL, 3. Sample concentration is 250 spores / mL; Figure 6 Sensitivity results of fluorescent quantitative PCR detection of Candida krusei (FAM channel); 1. Sample concentration is 1000 spores / mL, 2. Sample concentration is 500 spores / mL, 3. Sample concentration is 250 spores / mL; Figure 7 Sensitivity results of fluorescent quantitative PCR detection of Candida glabrata (VIC channel); 1. Sample concentration is 1000 spores / mL, 2. Sample concentration is 500 spores / mL, 3. Sample concentration is 250 spores / mL; Figure 8 Sensitivity results of fluorescent quantitative PCR detection of Candida tropicalis (CY5 channel); 1. Sample concentration is 1000 spores / mL, 2. Sample concentration is 500 spores / mL, 3. Sample concentration is 250 spores / mL; Fig. 9 Sensitivity results of fluorescent quantitative PCR detection of Candida parapsilosis (CY5 channel); 1. Sample concentration is 1000 spores / mL, 2. Sample concentration is 500 spores / mL, 3. Sample concentration is 250 spores / mL; Fig.10 Sensitivity experiment internal reference detection channel detection result diagram (ROX channel); 1. Sample concentration is 1000 spores / mL, 2. Sample concentration is 500 spores / mL, 3. Sample concentration is 250 spores / mL. DETAILED DESCRIPTION

[0018] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0019] The present invention relates to a multiple Candida nucleic acid detection kit and a detection method, which can simultaneously realize the combined qualitative detection of five pathogenic fungi, namely, Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis. Specific primers and specific probes of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis are used to detect the nucleic acid of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis in the presence of a heat-resistant DNA polymerase (Taq enzyme), high-quality deoxyribonucleoside triphosphates (dNTPs) and Mg. 2+ In the PCR reaction buffer, the polymerase chain reaction and fluorescent probe technology are combined to amplify and detect the highly conservative and specific sequences of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis, and the five bacteria are qualitatively detected. Thus, the purpose of rapid and real-time auxiliary diagnosis of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis infections is achieved.

[0020] Specific primers and specific probes designed for the ITS1, 5.8SrRNA and ITS2 gene sequences of Candida albicans; specific primers and specific probes designed for the ITS1, 5.8SrRNA and ITS2 gene sequences of Candida krusei; specific primers and specific probes designed for the ITS1, 5.8SrRNA and ITS2 gene sequences of Candida glabrata; specific primers and specific probes designed for the ITS1, 5.8SrRNA and ITS2 gene sequences of Candida tropicalis; specific primers and specific probes designed for the large subunit ribosomal RNA gene sequence of Candida parapsilosis.

[0021] Three fluorescent reporter genes can be used to mark different bacterial species. According to the primer characteristics of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis, Candida albicans and Candida krusei are grouped together, and the Candida albicans specific probe and the Candida krusei specific probe contain the first fluorescent reporter gene; Candida glabrata is grouped separately, and the Candida glabrata specific probe contains the second fluorescent reporter gene; Candida tropicalis and Candida parapsilosis are grouped together, and the Candida tropicalis specific probe and the Candida parapsilosis specific probe contain the third fluorescent reporter gene. The first fluorescent reporter gene, the second fluorescent reporter gene and the third fluorescent reporter gene are FAM, VIC and CY5 respectively; in addition, the Candida albicans specific probe, the Candida krusei specific probe, the Candida glabrata specific probe, the Candida tropicalis specific probe and the Candida parapsilosis specific probe all include MGB.

[0022] The specific sequence information is as follows: Candida albicans specific primer forward: SEQ ID No.1: 5'-GGGTTTGCTTGAAAGACGGTA-3' Candida albicans specific primer reverse: SEQ ID No.2: 5'-TTGAAGATATACGTGGTGGACGTTA--3' Candida albicans specific probe: 5'-FAM- Candida albicans probe sequence- MGB-3' Candida albicans probe sequence: SEQ ID No.3: 5'-ACCTAAGCCATTGTCAAAGCGATCC-3' Candida krusei specific primers forward: SEQ ID No.4: 5'-GCTGCGACTCGCCTGAA-3' Candida krusei specific primer reverse: SEQ ID No.5: 5'-TTGTCTCGCAACACTCGCTCT-3' Candida krusei specific probe: 5'-FAM-Candida krusei probe sequence- MGB-3' Candida krusei probe sequence: SEQ ID No.6: 5'-CTAGTTCGCTCGGCCAGCTTCG -3' Candida glabrata specific primer forward: SEQ ID No.7: 5'-ACGAGCAGCAGATTAATAGAGA-3' Candida glabrata specific primer reverse: SEQ ID No.8: 5'-TTGCGCCCTCTGGTATTC-3' Candida glabrata specific probe: 5'-VIC-Candida glabrata probe sequence-MGB-3' Candida glabrata probe sequence: SEQ ID No.9: 5'-TTGAGCGTCATTTCCTTCTCAAACAC -3' Forward primers specific for Candida tropicalis: SEQ ID No.10: 5'-GCGTCATTCTCCTCAA-3' Candida tropicalis specific primers reverse: SEQ ID No.11: 5'-AGTCGCTTAAAATAAGTTTCCA-3' Candida tropicalis-specific probe: 5'-CY5-Candida tropicalis probe sequence-MGB-3' Candida tropicalis probe sequence: SEQ ID No.12: 5'-CCTAGCGTATTGCTCAACACCAA -3' Candida parapsilosis specific primers forward: SEQ ID No.13: 5'-TGAGCGGTAGGATAAGTG-3' Candida parapsilosis specific primer reverse: SEQ ID No.14: 5'-TTATGCCAACATCCTAGGCC-3' Candida parapsilosis specific probe: 5'-CY5-Candida parapsilosis probe sequence-MGB-3' Candida parapsilosis probe sequence: SEQ ID No.15: 5'-AAGCCGCAGTCCTCAGTCTAAGCTG -3' When used for detection, it also includes the internal reference gene RNase P primers and probes, and the specific sequence information is as follows: Internal reference gene RNase P specific primer forward: SEQ ID No.16: 5'-AGATTTGGACCTGCGAGCG-3' Internal reference gene RNase P specific primer reverse: SEQ ID No.17: 5'-GAGCGGCTTGTCTCCACAAGT-3' Internal reference gene RNase P specific probe: 5'-ROX-RNase P probe sequence - MGB-3' Internal reference gene RNase P probe sequence: SEQ ID No.18: 5'-TTCTGACCTGAAGGCTCTGCG -3' The labeling groups of the five specific probes are FAM, VIC and CY5. The excitation wavelength of the FAM group is 492nm and the emission wavelength is 517nm; the excitation wavelength of the VIC group is 557nm and the emission wavelength is 574nm; the excitation wavelength of the CY5 group is 650nm and the emission wavelength is 670nm. There is no cross-overlapping section between the excitation wavelengths and emission wavelengths of the FAM, VIC and CY5 groups, so there is no mutual interference, thereby achieving simultaneous and specific detection of the five bacteria. The internal reference gene RNase P uses the ROX group.

[0023] The present invention also relates to a multiple Candida nucleic acid detection kit, comprising the above-mentioned primer probe set for detecting multiple Candida. The specific primers and specific probes of the five bacteria are used in the PCR system of the bacterial species detection sample, which can effectively detect whether the sample contains Candida albicans and / or Candida krusei and / or Candida glabrata and / or Candida tropicalis and / or Candida parapsilosis, and respectively perform qualitative analysis on the five Candida involved above.

[0024] Multiple Candida nucleic acid detection kits may include positive quality control products, negative quality control products, and detection reaction solutions; wherein the detection reaction solution includes Candida albicans specific primers, Candida albicans specific probes, Candida krusei specific primers, Candida krusei specific probes, Candida glabrata specific primers, Candida glabrata specific probes, Candida tropicalis specific primers, Candida tropicalis specific probes, Candida parapsilosis specific primers, and Candida parapsilosis specific probes. The detection reaction solution may also include internal reference gene RNaseP specific primers and probes, and the fluorescent dye in the internal reference gene RNase P specific probe is ROX, which is used for uniform correction of the intra-batch volume difference caused by pipetting error or evaporation, and correction of signal changes caused by foam or evaporation and condensation in a single reaction run.

[0025] In certain embodiments of the present invention, the positive quality control products and the detection reaction solution in the multiple Candida nucleic acid detection kit can be provided in the form of freeze-dried powder. Wherein the positive quality control product (freeze-dried powder) is composed of a solution containing RNase gene, a solution containing Candida albicans gene fragment, a Candida krusei gene fragment solution, a Candida glabrata gene fragment solution, a Candida tropicalis gene fragment solution, a Candida parapsilosis gene fragment solution and a freeze-dried protective agent. It is necessary to operate in a clean bench during preparation. The negative quality control product is sterile enzyme-free deionized water. The detection reaction solution includes Candida albicans specific primers, Candida albicans specific probes, Candida krusei specific primers, Candida krusei specific probes, Candida glabrata specific primers, Candida glabrata specific probes, Candida tropicalis specific primers, Candida tropicalis specific probes, Candida parapsilosis specific primers, Candida parapsilosis specific probes and internal reference gene RNaseP specific primers and probes.

[0026] When in use, in a clean bench, the freeze-dried powder positive control product is prepared into a solution with sterile enzyme-free deionized water (or negative control product), and then prepared with the sample to be tested to form a fluorescent PCR reaction system for fluorescent PCR amplification reaction. The Candida sample is qualitatively determined based on whether the characteristic peak of the fluorescent reporter gene appears and the position of the characteristic peak.

[0027] The Candida albicans-specific probe and the Candida krusei-specific probe contain a first fluorescent reporter gene; in the first fluorescent reporter gene color development channel, if the Tm value of the characteristic peak is between 71.5 and 74.5°C, it is determined to be Candida albicans positive; if the Tm value is between 78.3 and 80.7°C, it is determined to be Candida krusei positive; if there is no characteristic peak or the Tm value is not between 71.5 and 74.5°C and 78.3 and 80.7°C, it is negative for Candida albicans and Candida krusei; the Candida glabrata-specific probe contains a second fluorescent reporter gene; in the second fluorescent reporter gene color development channel, if the Tm value of the characteristic peak is between 72.5 and 74. If the Tm value is between 70.2 and 72.1°C, it is determined to be positive for Candida glabrata; if there is no characteristic peak or the Tm value is not between 72.5 and 74.7°C, it is negative for Candida glabrata; the Candida tropicalis-specific probe and the Candida parapsilosis-specific probe contain a third fluorescent reporter gene, and in the second fluorescent reporter gene color development channel, if the Tm value of the characteristic peak is between 70.2 and 72.1°C, it is determined to be positive for Candida tropicalis; if the Tm value is between 76.5 and 78.7°C, it is determined to be positive for Candida parapsilosis; if there is no characteristic peak or the Tm value is not between 70.2 and 72.1°C and 76.5 and 78.7°C, it is negative for Candida tropicalis and negative for Candida parapsilosis.

[0028] Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis belong to the same genus, and have high sequence similarity. In order to distinguish the five bacteria, it is difficult to design the primers that can be used. Through the above primers and probes, it is possible to detect with high sensitivity whether Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis exist in the sample to be tested. In the same detection process, Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis are qualitatively analyzed respectively. The kit has the characteristics of high sensitivity and high efficiency.

[0029] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0030] Example 1: Preparation of the kit 1.1 Design and synthesis of primers and probes The specific primers and probes are designed for the ITS1, 5.8SrRNA and ITS2 gene sequences of Candida albicans; the specific primers and probes are designed for the ITS1, 5.8SrRNA and ITS2 gene sequences of Candida krusei; the specific primers and probes are designed for the ITS1, 5.8SrRNA and ITS2 gene sequences of Candida glabrata; the specific primers and probes are designed for the ITS1, 5.8SrRNA and ITS2 gene sequences of Candida tropicalis; the specific primers and probes are designed for the large subunit ribosomal RNA gene sequence of Candida parapsilosis. The reporter groups for the labeling of the five specific probes are also selected as FAM, VIC and CY5, respectively, and the quenching group is MGB, and the sequence data are all from GeneBank. The primers and probes are synthesized by General Biotech. The specific primer sequences are as follows: Candida albicans specific primer forward: SEQ ID No.1: 5'-GGGTTTGCTTGAAAGACGGTA-3' Candida albicans specific primer reverse: SEQ ID No.2: 5'-TTGAAGATATACGTGGTGGACGTTA--3' Candida albicans specific probe: 5'-FAM- Candida albicans probe sequence- MGB-3' Candida albicans probe sequence: SEQ ID No.3: 5'-ACCTAAGCCATTGTCAAAGCGATCC-3' Candida krusei specific primers forward: SEQ ID No.4: 5'-GCTGCGACTCGCCTGAA-3' Candida krusei specific primer reverse: SEQ ID No.5: 5'-TTGTCTCGCAACACTCGCTCT-3' Candida krusei specific probe: 5'-FAM-Candida krusei probe sequence- MGB-3' Candida krusei probe sequence: SEQ ID No.6: 5'-CTAGTTCGCTCGGCCAGCTTCG -3' Candida glabrata specific primer forward: SEQ ID No.7: 5'-ACGAGCAGCAGATTAATAGAGA-3' Candida glabrata specific primer reverse: SEQ ID No.8: 5'-TTGCGCCCTCTGGTATTC-3' Candida glabrata specific probe: 5'-VIC-Candida glabrata probe sequence-MGB-3' Candida glabrata probe sequence: SEQ ID No.9: 5'-TTGAGCGTCATTTCCTTCTCAAACAC -3' Forward primers specific for Candida tropicalis: SEQ ID No.10: 5'-GCGTCATTCTCCTCAA-3' Candida tropicalis specific primers reverse: SEQ ID No.11: 5'-AGTCGCTTAAAATAAGTTTCCA-3' Candida tropicalis-specific probe: 5'-CY5-Candida tropicalis probe sequence-MGB-3' Candida tropicalis probe sequence: SEQ ID No.12: 5'-CCTAGCGTATTGCTCAACACCAA -3' Candida parapsilosis specific primers forward: SEQ ID No.13: 5'-TGAGCGGTAGGATAAGTG-3' Candida parapsilosis specific primer reverse: SEQ ID No.14: 5'-TTATGCCAACATCCTAGGCC-3' Candida parapsilosis specific probe: 5'-CY5-Candida parapsilosis probe sequence-MGB-3' Candida parapsilosis probe sequence: SEQ ID No.15: 5'-AAGCCGCAGTCCTCAGTCTAAGCTG -3' In certain embodiments of the present invention, primers and probes for the internal reference gene RNase P are also included, and the specific sequence information is as follows: Internal reference gene RNase P specific primer forward: SEQ ID No.16: 5'-AGATTTGGACCTGCGAGCG-3' Internal reference gene RNase P specific primer reverse: SEQ ID No.17: 5'-GAGCGGCTTGTCTCCACAAGT-3' Internal reference gene RNase P specific probe: 5'-ROX-RNase P probe sequence - MGB-3' Internal reference gene RNase P probe sequence: SEQ ID No.18: 5'-TTCTGACCTGAAGGCTCTGCG -3' 1.2 Preparation of positive and negative quality control products The positive quality control product includes a solution containing RNase gene, a solution containing genomes of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis, and a lyophilization protective agent. The above materials are mixed evenly and prepared into lyophilized powder; the negative quality control product is sterile enzyme-free deionized water.

[0031] When in use, in an ultra-clean workbench, prepare a solution of the positive control substance in the freeze-dried powder state using sterile, enzyme-free deionized water (or negative control substance).

[0032] 1.3 Fluorescence PCR reaction solution composition

[0033] The primers and probes synthesized in step 1.1 are added to the reaction solution system, and the template is the sample to be detected.

[0034] 1.4 Kit Composition The kit consists of detection reaction solution (lyophilized powder), positive quality control product (lyophilized powder), and negative quality control product (sterile enzyme-free deionized water).

[0035] Example 2: Using a multi-candida nucleic acid detection kit to detect the presence of Candida in a sample 2.1 Sample processing Sample extraction: First dissolve the positive quality control product (lyophilized powder) with sterile enzyme-free deionized water, and then use the nucleic acid extraction kit to extract nucleic acid from the sample to be tested.

[0036] 2.2 Reagent preparation Determine the number of reaction tubes N (number of samples + 1 negative control tube + 1 positive control tube), and use sterile, enzyme-free deionized water solution to test the reaction solution (lyophilized powder), and dispense 19 μL per tube into suitable PCR reaction tubes.

[0037] 2.3 Sample testing 6 μL of nucleic acid extraction solution of the sample to be tested, positive quality control, and negative quality control were added to the PCR reaction tubes, the PCR reaction tube caps were tightly closed, and the liquid on the tube wall was centrifuged to the bottom of the tube instantly, and the PCR amplification reaction was immediately performed. The real-time fluorescence PCR reaction was performed using an ABI7500 real-time fluorescence PCR instrument, using the real-time data acquisition mode.

[0038] 2.4 Reaction procedure Detection channel settings: For Candida gene detection, select FAM, VIC, and CY5 channels, select the ROX channel for internal standard, and select "None" for reference fluorescence. Set the positive control (Positive Control), negative control (Negative Control), and sample (Unknown) in the corresponding order of the samples, and set the sample name and position.

[0039] The fluorescence PCR amplification reaction procedure is shown in the following table:

[0040] 2.5 Result judgment method The target detection signals are: FAM, VIC, and CY5 channels, and the internal reference detection signal is the ROX channel.

[0041] Determination of positive and negative results: First, analyze whether the internal reference detects a characteristic peak of the melting curve in the ROX channel, and whether the Tm value is between 69.7 and 71.2 °C. If yes, it means that this test is valid and subsequent analysis can be continued: 1) If the FAM channel detects a characteristic peak of Tm (71.5~74.5℃), it means that the test result for Candida albicans is positive; if the FAM channel detects a characteristic peak of Tm (78.3~80.7℃), it means that the test result for Candida krusei is positive. If there is no characteristic peak or Tm is not between 71.5~74.5℃ and 78.3~80.7℃, it is negative for Candida albicans or Candida krusei.

[0042] 2) If the VIC channel detects a characteristic peak of Tm (72.5~74.7℃), it means that the test result of Candida glabrata is positive; if there is no characteristic peak or Tm is not between 72.5~74.7℃, it is negative for Candida glabrata.

[0043] 3) If the CY5 channel detects the characteristic peak of Tm (70.2~72.1℃), it means that the test result of Candida tropicalis is positive; if the CY5 channel detects the characteristic peak of Tm (76.5~78.7℃), it means that the test result of Candida parapsilosis is positive. If there is no characteristic peak or Tm is not between 0.2~72.1℃ and 76.5~78.7℃, it is negative for Candida tropicalis and Candida parapsilosis.

[0044] The identification methods of various bacterial species can be shown in Table 1.

[0045] Table 1 Test result judgment method

[0046] If there are no obvious characteristic peaks in the melting curves of the FAM, VIC, CY5 and ROX channels, the causes and suggestions are as follows: (1) unreasonable sample collection, transportation and processing, and the target bacteria content in the sample is too low. It is recommended to resample and send for testing; (2) abnormal nucleic acid extraction process leads to nucleic acid loss. It is recommended to re-extract nucleic acid.

[0047] If there are substances in the sample that inhibit the PCR reaction, the sample can be diluted and tested again.

[0048] Example 3: Study on the specificity of multiple Candida nucleic acid detection kits The nucleic acid extraction or purification kit extracts the genomic DNA of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, Candida parapsilosis and other common clinical pathogens, and then amplifies according to the fluorescent PCR reaction system and reaction procedure to detect the specificity of the primer probe of the kit. The 10 common clinical pathogens selected are as follows: Cryptococcus neoformans, Rhizopus oryzae, Fusarium moniliforme, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Talaromyces marneffei, Acinetobacter baumannii, Aspergillus fumigatus and Aspergillus flavus. The samples to be tested containing the above-mentioned species were tested according to the detection method of Example 2, and the results are as follows. Figure 1-Figure 4 shown.

[0049] like Figure 4 As shown in Figure 1, all sample internal reference channels have normal peaks, and there is no false negative in the test results. Figure 1 As shown in Figure 2, the melting curves of Candida albicans and Candida krusei have characteristic peaks, while the melting curves of the other 10 bacteria have no obvious characteristic peaks. Figure 2As shown in Figure 2, the melting curve of Candida glabrata has a characteristic peak, while the melting curves of the other 10 bacteria have no obvious characteristic peaks. Figure 3 As shown in the figure, the melting curves of Candida tropicalis and Candida parapsilosis have characteristic peaks, while the melting curves of the other 10 bacteria have no obvious characteristic peaks. From the above results, it can be seen that the primers and probes of this kit have high specificity.

[0050] Example 4: Study on the sensitivity of multiple Candida nucleic acid detection kits The bacterial suspensions of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis were diluted to 1000 spores / mL, 500 spores / mL and 250 spores / mL, respectively. Genomic DNA was extracted using a nucleic acid extraction kit, and fluorescent PCR amplification and detection were performed according to the steps of Example 2. The results are as follows: Figure 5-Figure 9 shown.

[0051] First, Fig.10 As shown in the figure, all sample internal reference channels have normal peaks, and there are no false negative results in the test. Figure 5 This is the detection of Candida albicans. Obvious peaks can be seen at 1000 spores / mL, 500 spores / mL, and 250 spores / mL. Figure 6 For the detection of Candida krusei, Figure 7 For the detection of Candida glabrata, Figure 8 For the detection of Candida tropicalis, Fig. 9 The results of the Candida parapsilosis test are as follows: obvious peaks can be seen in the samples tested at 1000 spores / mL, 500 spores / mL, and 250 spores / mL, indicating that the kit can detect a concentration of 250 spores / mL for Candida parapsilosis, and that the kit has high sensitivity.

[0052] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made according to the scope of application of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A primer probe set for detecting multiple Candida species, characterized in that: The method comprises a specific forward primer, a specific reverse primer and a specific probe for detecting at least two species of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis respectively; Among them, the specific forward primer, specific reverse primer and specific probe sequences for detecting Candida albicans are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 respectively; The specific forward primer, specific reverse primer and specific probe sequences for detecting Candida krusei are shown in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 respectively; The specific forward primer, specific reverse primer and specific probe sequences for detecting Candida glabrata are shown in SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9 respectively; The specific forward primer, specific reverse primer and specific probe sequences for detecting Candida tropicalis are shown in SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12 respectively; The specific forward primer, specific reverse primer and specific probe sequences for detecting Candida parapsilosis are shown in SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No. 15 respectively.

2. The primer probe set for detecting multiple Candida species according to claim 1, characterized in that: The Candida albicans specific probe and the Candida krusei specific probe contain a first fluorescent reporter gene; the Candida glabrata specific probe contains a second fluorescent reporter gene; The Candida tropicalis-specific probe and the Candida parapsilosis-specific probe contain a third fluorescent reporter gene.

3. The primer probe set for detecting multiple Candida species according to claim 2, characterized in that: The first fluorescent reporter gene, the second fluorescent reporter gene and the third fluorescent reporter gene are FAM, VIC and CY5 respectively; the Candida albicans specific probe, the Candida krusei specific probe, the Candida glabrata specific probe, the Candida tropicalis specific probe and the Candida parapsilosis specific probe all include MGB.

4. The primer probe set for detecting multiple Candida species according to any one of claims 1 to 3, characterized in that: The method comprises specific forward primers, specific reverse primers and specific probes for detecting Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and Candida parapsilosis respectively.

5. A multiple Candida nucleic acid detection kit, characterized in that: A primer probe set for detecting multiple Candida species as described in any one of claims 1 to 4.

6. The multiple Candida nucleic acid detection kit according to claim 5, characterized in that: It also includes a positive quality control product, a negative quality control product and a detection reaction solution, wherein the detection reaction solution includes a primer probe set; Preferably, the positive quality control product includes artificial simulated alveolar lavage fluid, a solution containing the genome of Candida albicans, a solution containing the genome of Candida krusei, a solution containing the genome of Candida glabrata, a solution containing the genome of Candida tropicalis, and a solution containing the genome of Candida parapsilosis; the negative quality control product is sterile enzyme-free deionized water.

7. The multiple Candida nucleic acid detection kit according to claim 6, characterized in that: It also includes a specific forward primer, a specific reverse primer and a specific probe for detecting the internal reference gene RNase P, and the sequences are shown in SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18 respectively.

8. A method for detecting multiple types of Candida species, characterized in that: The detection is performed using the primer probe set described in any one of claims 1 to 4, or the detection is performed using the kit described in any one of claims 5 to 7.

9. The method for detecting multiple types of Candida according to claim 8, characterized in that: The Candida albicans in the sample is qualitatively identified based on whether the characteristic peak of the fluorescent reporter gene appears and the position of the characteristic peak.

10. The method for detecting multiple types of Candida according to claim 9, characterized in that: The Candida albicans specific probe and the Candida krusei specific probe contain a first fluorescent reporter gene; in the first fluorescent reporter gene color development channel, if the Tm value of the characteristic peak is between 71.5 and 74.5°C, it is determined to be Candida albicans positive; If the Tm value is between 78.3 and 80.7°C, it is judged as positive for Candida krusei; if there is no characteristic peak or the Tm value is not between 71.5 and 74.5°C and 78.3 and 80.7°C, it is negative for Candida albicans and negative for Candida krusei; The Candida glabrata specific probe contains a second fluorescent reporter gene; in the second fluorescent reporter gene color development channel, if the Tm value of the characteristic peak is between 72.5 and 74.7°C, it is determined to be Candida glabrata positive; if there is no characteristic peak or the Tm value is not between 72.5 and 74.7°C, it is determined to be Candida glabrata negative; The Candida tropicalis-specific probe and the Candida parapsilosis-specific probe contain a third fluorescent reporter gene. In the second fluorescent reporter gene color development channel, if the Tm value of the characteristic peak is between 70.2 and 72.1°C, it is determined to be positive for Candida tropicalis. If the Tm value is between 76.5 and 78.7°C, it is judged to be positive for Candida parapsilosis; if there is no characteristic peak or the Tm value is not between 70.2 to 72.1°C and 76.5 to 78.7°C, it is negative for Candida tropicalis and Candida parapsilosis.

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