A primer-probe composition, a kit and a preparation method thereof for detecting pathogens of chromoblastomycosis

Abstract

The invention provides a primer-probe composition, a kit and a preparation method thereof for detecting the pathogen of chromoblastomycosis, and belongs to the technical field of biological detection. The primer-probe composition comprises an upstream primer, a downstream primer and a fluorescent probe designed for Helminthosporium; an upstream primer, a downstream primer and a fluorescent probe designed for Cladosporium; an upstream primer, a downstream primer and a fluorescent probe designed for Ochrasporium; an upstream primer, a downstream primer and a fluorescent probe designed for Cladosporium; an upstream primer, a downstream primer and a fluorescent probe designed for Vanadium; and an upstream primer, a downstream primer and a fluorescent probe designed as an internal reference for a human RNase P gene. The invention adopts a multiplex fluorescent PCR technology combined with a freeze-drying technology, is easy to operate, overcomes the false positive phenomenon, and has high sensitivity and strong specificity, and can simultaneously detect the infection conditions of five pathogens causing chromoblastomycosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a primer-probe composition, a kit and a preparation method thereof for detecting a pathogen of chromoblastomycosis. Background Art

[0002] Chromoblastomycosis is a specific infection of the skin caused by one of several dark-colored fungi (pigmented fungi) that results in the production of sclerotic masses in tissues, affecting mostly immunocompetent people in tropical and subtropical regions. It is characterized by papilloma-like nodules with a tendency to ulcerate, with ulcerated nodules appearing on exposed parts of the body. Chromoblastomycosis is caused by pathogens from the genera Bipolaris, Cladosporium, Ochronosis, Rhinocladiella, and Wangiella.

[0003] At present, the detection of the pathogen of chromoblastomycosis is mainly carried out by microscopy, isolation culture and histopathology, but the above methods are complicated, time-consuming and have poor sensitivity, which makes it difficult to meet the needs of immediate detection of pathogens. Molecular diagnosis is an emerging and useful tool for identifying the molecular composition of certain fungal infections. The development of accurate molecular diagnostic technology for the diagnosis and treatment of the pathogen of chromoblastomycosis is of vital importance for timely diagnosis and guidance of medication. Summary of the invention

[0004] In view of the above-mentioned technical defects, the present invention provides a primer-probe composition, a kit and a preparation method thereof for detecting the pathogen of chromoblastomycosis. Based on the fluorescent quantitative PCR detection technology, five pathogens of the genera of Helminthosporium, Cladosporium, Ochrasporium, Cladosporium and Vancomycetes can be simultaneously detected by one reaction, and it has the advantages of high sensitivity, strong specificity, short detection time and high detection throughput.

[0005] The technical solution adopted by the present invention is:

[0006] A primer-probe composition for detecting a pathogen of chromoblastomycosis comprises:

[0007] SEQ ID NO.1: An upstream primer designed for Helminthosporium, the sequence of which is 5'-ACTTGCTTGCAGTTGCTCATCC-3'; referred to as Bipolaris-F;

[0008] SEQ ID NO.2: A downstream primer designed for Helminthosporium, the sequence of which is 5'-CCCGAAGAGAGGTACGTGACAG-3'; referred to as Bipolaris-R;

[0009] SEQ ID NO.3: A fluorescent probe designed for Helminthosporium, the sequence of which is 5'-ACCTTTATCCCACCGCCCAAACTGATGCTG-3'; referred to as Bipolaris-P; the 5' end of the fluorescent probe is labeled with a reporter group 6-FAM, and the 3' end is labeled with a quencher group BHQ1;

[0010] SEQ ID NO.4: an upstream primer designed for Cladosporium, the sequence of which is 5'-GGACACTTCAAACTCTTGCGTAAC-3'; referred to as Cladosporium-F;

[0011] SEQ ID NO.5: A downstream primer designed for Cladosporium, the sequence of which is 5'-CATTTCGCTGCGTTCTTCATCG-3'; referred to as Cladosporium-R;

[0012] SEQ ID NO.6: A fluorescent probe designed for Cladosporium, the sequence of which is 5'-CCAGAACCAAGAGAT-3'; referred to as Cladosporium-P; the 5' end of the fluorescent probe is labeled with a reporter group ROX, and the 3' end is labeled with a quencher group MGB;

[0013] SEQ ID NO.7: An upstream primer designed for Ochrosporum, the sequence of which is 5'-GGACCAAGGAGTCTAACATCTATGC-3'; referred to as Ochronosis-F;

[0014] SEQ ID NO.8: A downstream primer designed for Ochrosporum, the sequence of which is 5'-GTCGATGGTGCGTCCTGTAAC-3'; referred to as Ochronosis-R;

[0015] SEQ ID NO.9: A fluorescent probe designed for Ochrosporum, the sequence of which is 5'-ACTCCCACCTCCGTTCACTTTCATTGCG-3'; referred to as Ochronosis-P; the 5' end of the fluorescent probe is labeled with a reporter group CY5, and the 3' end is labeled with a quencher group BHQ3;

[0016] SEQ ID NO.10: An upstream primer designed for Rhinocladiella, the sequence of which is 5'-GTTGTCAGCGACCCGTCTTG-3'; referred to as Rhinocladiella-F;

[0017] SEQ ID NO.11: A downstream primer designed for Rhinocladiella, the sequence of which is 5'-TCCTACCTCCGTTCACTTTCATTTC-3'; referred to as Rhinocladiella-R;

[0018] SEQ ID NO.12: A fluorescent probe designed for the genus Rhinocladiella, the sequence of which is 5'-ACACGGACCAAGGAGTCTAACATCTATGCGAG-3'; referred to as Rhinocladiella-P; the 5' end of the fluorescent probe is labeled with a reporter group 6-FAM, and the 3' end is labeled with a quencher group BHQ1;

[0019] SEQ ID NO.13: An upstream primer designed for Wangiella, the sequence of which is 5'-GGAGAGGGAGCCTGAGAAACG-3'; referred to as Wangiella-F;

[0020] SEQ ID NO.14: A downstream primer designed for Wangiella, the sequence of which is 5'-TCATTCCAATTACGAGACCCGAAAG-3'; referred to as Wangiella-R;

[0021] SEQ ID NO.15: A fluorescent probe designed for Wangiella, the sequence of which is 5'-TACCACATCCAAGGAAGGCAGCAGGCG-3'; referred to as Wangiella-P; the 5' end of the fluorescent probe is labeled with a reporter group ROX, and the 3' end is labeled with a quencher group BHQ2;

[0022] SEQ ID NO.16: An upstream primer designed for human RNase P gene, the sequence of the upstream primer is 5'-AGATTTGGACCTGCGAGCG-3'; referred to as IC-F;

[0023] SEQ ID NO.17: A downstream primer designed for human RNase P gene, the sequence of the downstream primer is 5'-GAGCGGCTGTCTCCACAAGT-3'; referred to as IC-R;

[0024] SEQ ID NO.18: A fluorescent probe designed for human RNase P gene, the sequence of the fluorescent probe is 5'-TTCTGACCTGAAGGCTCTGCGCG-3'; abbreviated as IC-P; the 5' end of the fluorescent probe is labeled with a reporter group VIC, and the 3' end is labeled with a quencher group BHQ1.

[0025] The primer probe introduced into the present invention is used to monitor the collection, transportation, extraction and amplification process of the sample to be tested, possible reaction inhibitors, and avoid false negatives, which is suitable for the diagnosis of the pathogen of chromoblastomycosis.

[0026] A kit for detecting the pathogen of chromoblastomycosis comprises a freeze-dried PCR reaction system, a freeze-dried positive control and a negative control.

[0027] The PCR reaction system includes a tube A PCR reaction system and a tube B PCR reaction system. The tube A PCR reaction system and the tube B PCR reaction system are prepared according to 20 μL per person, 8 μL of lyophilized protective agent is added to each person, and the two tubes A and B are divided into two PCR reaction tubes.

[0028] The two packaged PCR reaction systems were subjected to the following freeze-drying procedure: pre-freezing stage: pre-freezing at -45°C for 4 hours; main drying stage: evacuating the vacuum to 1-10Pa in 5 minutes, raising the shelf temperature to -40°C in 5 minutes; then keeping at -40°C for 10 hours; analytical drying stage: maintaining the vacuum at 1-10Pa, raising the shelf temperature to 30°C in 6.5 hours; then keeping at 30°C for 2 hours to obtain the freeze-dried PCR reaction system.

[0029] The mass percentage concentrations of the components in the freeze-dried protective agent are: 5% polysucrose 400, 5% xylitol, 2% glutamic acid, and 1% vitamin E.

[0030] The positive control is a plasmid mixture of an artificial synthetic plasmid of the genus Helminthosporium, an artificial synthetic plasmid of the genus Cladosporium, an artificial synthetic plasmid of the genus Ochrasporium, an artificial synthetic plasmid of the genus Cladosporium, an artificial synthetic plasmid of the genus Vanadium and an internal reference artificial synthetic plasmid, and the concentration ratio of the above artificial synthetic plasmids is 1:1:1:1:1:1:1.

[0031] The negative control is TE Buffer.

[0032] The internal reference artificial synthetic plasmid is a plasmid into which a human RNase P gene sequence fragment is inserted, and is used to monitor the collection, transportation, extraction and amplification process of the sample to be tested to avoid false negative results of the sample.

[0033] A method for preparing a kit for detecting the pathogen of chromoblastomycosis comprises the following steps: preparing a PCR reaction system, preparing a positive control and a negative control, freeze-drying the PCR reaction system and the positive control, assembling a detection kit and constructing a reaction program.

[0034] The reaction procedure of the construction: the reaction procedure includes decontamination at 37°C for 5 minutes; pre-denaturation at 95°C for 5 minutes; amplification reaction: reaction at 95°C for 15 seconds, reaction at 60°C for 30 seconds, and 40 cycles.

[0035] During the cycling step, fluorescence was detected at 60°C, and fluorescence signals of FAM, ROX, CY5, and VIC channels were collected.

[0036] The beneficial effects of the present invention are:

[0037] The present invention adopts multiplex fluorescence PCR technology combined with freeze-drying technology, which is not only easy to operate and overcomes the false positive phenomenon, but also has high sensitivity and strong specificity. It can simultaneously detect the infection of five pathogens that cause chromoblastomycosis, meets the development needs of clinical detection of chromoblastomycosis, and can guide medication as soon as possible; the reagent can be stored and transported at room temperature, the enzyme activity can be effectively preserved, and no liquid preparation and packaging steps are required. 25 μL of target can be directly added to react, and the detection cycle is short, and the detection can be completed within 1.5 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 1×10 3 -1×10 7 PCR amplification curve of Helminthosporium detection at 100 copies / mL and the fluorescence quantitative standard curve of the kit of the present invention for Helminthosporium detection;

[0039] Figure 2 1×10 3 -1×10 7 PCR amplification curve of Cladosporium detection at 100 copies / mL and the fluorescence quantitative standard curve of Cladosporium detection by the kit of the present invention;

[0040] Figure 3 1×10 3 -1×10 7 PCR amplification curve of Ochra detection at 100 copies / mL and fluorescence quantitative standard curve of Ochra detection by the kit of the present invention;

[0041] Figure 4 1×10 3 -1×10 7 PCR amplification curve of Cladosporium detection at 100 copies / mL and the fluorescence quantitative standard curve of Cladosporium detection by the kit of the present invention;

[0042] Figure 5 1×10 3 -1×10 7 PCR amplification curve of Wannella detection at 100 copies / mL and the fluorescence quantitative standard curve of the kit of the present invention for Cladosporium detection;

[0043] Figure 6It is a comparison chart of PCR amplification curves of Helminthosporium, Cladosporium, Ochrasporium, Cladosporium, Vancomyces, Epidermophyton, Trichophyton, Microsporum, Malassezia furfur, Trichoderma nigra and Trichoderma albus;

[0044] Figure 7 The figure is a graph showing the test results of 13 clinical samples using the present invention. DETAILED DESCRIPTION

[0045] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the present invention, those skilled in the art can make various technical changes or modifications to the present invention through technical attempts, and these equivalent forms generally fall within the scope defined by the appended claims of the present application.

[0046] Example 1 A primer combination for detecting the pathogen of chromoblastomycosis

[0047] The gene sequences of Helminthosporium, Cladosporium, Ochrasporium, Cladosporium, Vanadium and human RNase P were obtained from the NCBI GeneBank database, and the gene sequences were compared to design the following primers and probes:

[0048] SEQ ID NO.1: An upstream primer designed for Helminthosporium, the sequence of which is 5'-ACTTGCTTGCAGTTGCTCATCC-3'; referred to as Bipolaris-F;

[0049] SEQ ID NO.2: A downstream primer designed for Helminthosporium, the sequence of which is 5'-CCCGAAGAGAGGTACGTGACAG-3'; referred to as Bipolaris-R;

[0050] SEQ ID NO.3: A fluorescent probe designed for Helminthosporium, the sequence of which is 5'-ACCTTTATCCCACCGCCCAAACTGATGCTG-3'; referred to as Bipolaris-P; the 5' end of the fluorescent probe is labeled with a reporter group 6-FAM, and the 3' end is labeled with a quencher group BHQ1;

[0051] SEQ ID NO.4: an upstream primer designed for Cladosporium, the sequence of which is 5'-GGACACTTCAAACTCTTGCGTAAC-3'; referred to as Cladosporium-F;

[0052] SEQ ID NO.5: A downstream primer designed for Cladosporium, the sequence of which is 5'-CATTTCGCTGCGTTCTTCATCG-3'; referred to as Cladosporium-R;

[0053] SEQ ID NO.6: A fluorescent probe designed for Cladosporium, the sequence of which is 5'-CCAGAACCAAGAGAT-3'; referred to as Cladosporium-P; the 5' end of the fluorescent probe is labeled with a reporter group ROX, and the 3' end is labeled with a quencher group MGB;

[0054] SEQ ID NO.7: An upstream primer designed for Ochrosporum, the sequence of which is 5'-GGACCAAGGAGTCTAACATCTATGC-3'; referred to as Ochronosis-F;

[0055] SEQ ID NO.8: A downstream primer designed for Ochrosporum, the sequence of which is 5'-GTCGATGGTGCGTCCTGTAAC-3'; referred to as Ochronosis-R;

[0056] SEQ ID NO.9: A fluorescent probe designed for Ochrosporum, the sequence of which is 5'-ACTCCCACCTCCGTTCACTTTCATTGCG-3'; referred to as Ochronosis-P; the 5' end of the fluorescent probe is labeled with a reporter group CY5, and the 3' end is labeled with a quencher group BHQ3;

[0057] SEQ ID NO.10: An upstream primer designed for Rhinocladiella, the sequence of which is 5'-GTTGTCAGCGACCCGTCTTG-3'; referred to as Rhinocladiella-F;

[0058] SEQ ID NO.11: A downstream primer designed for Rhinocladiella, the sequence of which is 5'-TCCTACCTCCGTTCACTTTCATTTC-3'; referred to as Rhinocladiella-R;

[0059] SEQ ID NO.12: A fluorescent probe designed for the genus Rhinocladiella, the sequence of which is 5'-ACACGGACCAAGGAGTCTAACATCTATGCGAG-3'; referred to as Rhinocladiella-P; the 5' end of the fluorescent probe is labeled with a reporter group 6-FAM, and the 3' end is labeled with a quencher group BHQ1;

[0060] SEQ ID NO.13: An upstream primer designed for Wangiella, the sequence of which is 5'-GGAGAGGGAGCCTGAGAAACG-3'; referred to as Wangiella-F;

[0061] SEQ ID NO.14: A downstream primer designed for Wangiella, the sequence of which is 5'-TCATTCCAATTACGAGACCCGAAAG-3'; referred to as Wangiella-R;

[0062] SEQ ID NO.15: A fluorescent probe designed for Wangiella, the sequence of which is 5'-TACCACATCCAAGGAAGGCAGCAGGCG-3'; referred to as Wangiella-P; the 5' end of the fluorescent probe is labeled with a reporter group ROX, and the 3' end is labeled with a quencher group BHQ2;

[0063] SEQ ID NO.16: An upstream primer designed for human RNase P gene, the sequence of the upstream primer is 5'-AGATTTGGACCTGCGAGCG-3'; referred to as IC-F;

[0064] SEQ ID NO.17: A downstream primer designed for human RNase P gene, the sequence of the downstream primer is 5'-GAGCGGCTGTCTCCACAAGT-3'; referred to as IC-R;

[0065] SEQ ID NO.18: A fluorescent probe designed for human RNase P gene, the sequence of the fluorescent probe is 5'-TTCTGACCTGAAGGCTCTGCGCG-3'; abbreviated as IC-P; the 5' end of the fluorescent probe is labeled with a reporter group VIC, and the 3' end is labeled with a quencher group BHQ1.

[0066] The above primers and probe plasmids were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0067] Example 2 A method for preparing a kit for detecting the pathogen of chromoblastomycosis

[0068] Step 1: Prepare PCR reaction system

[0069] The PCR reaction system includes a tube A PCR reaction system and a tube B PCR reaction system. The primer composition provided in Example 1 is used to prepare reagents. The tube A PCR reaction system is prepared according to Table 1, and the tube B PCR reaction system is prepared according to Table 2.

[0070] Table 1 Components and dosages of PCR reaction system in tube A

[0071]

[0072] Table 2 Components and dosages of tube B PCR reaction system

[0073]

[0074] GUeasy Taq DNA polymerase was used as the reaction enzyme, and the antibody modification buffer (Mg 2+ ) was used as reaction buffer, Taq DNA polymerase was purchased from General Biotechnology (Anhui) Co., Ltd., and UDG enzyme was purchased from Nanjing Novozymes Biotech Co., Ltd.

[0075] Step 2: Prepare positive and negative controls

[0076] Positive control: The target gene sequence of Helminthosporium, the target gene sequence of Cladosporium, the target gene sequence of Ochrasporium, the target gene sequence of Cladosporium, the target gene sequence of Vanadium, and the human RNase P gene sequence were respectively inserted into the pUC57 vector plasmid, and after expanded culture, identification and plasmid extraction, the artificial synthetic plasmid of Helminthosporium, the artificial synthetic plasmid of Cladosporium, the artificial synthetic plasmid of Ochrasporium, the artificial synthetic plasmid of Cladosporium, the artificial synthetic plasmid of Vanadium, and the internal reference artificial synthetic plasmid were obtained.

[0077] The above synthetic plasmids were quantified to 6×10 7 copies / mL, and then mixed in a volume ratio of 1:1:1:1:1:1 to prepare a positive control;

[0078] The negative control was TE Buffer.

[0079] Step 3: PCR reaction system and positive control freeze-drying

[0080] (1) Prepare the lyophilization protective agent according to the following components and mass percentage concentrations: 5% polysucrose 400, 5% xylitol, 2% glutamic acid, and 1% vitamin E.

[0081] (2) Prepare PCR reaction system A and PCR reaction system B according to 20 μL per person, add 8 μL of the above-mentioned lyophilization protective agent to each person, and divide them into two PCR reaction tubes A and B, 28 μL per tube, and place the two divided PCR reaction systems in a vacuum freeze dryer. The following freeze-drying procedures are used: pre-freezing stage: pre-freeze at -45℃ for 4 hours; main drying stage: vacuumize for 5 minutes until the vacuum degree drops to 1-10Pa, and increase the shelf temperature to -40℃ for 5 minutes; then keep it at -40℃ for 10 hours; analytical drying stage: maintain the vacuum degree at 1-10Pa, and increase the shelf temperature to 30℃ for 6.5 hours; then keep it at 30℃ for 2 hours to obtain the freeze-dried PCR reaction system.

[0082] (3) Insert the target gene sequence of Helminthosporium, the target gene sequence of Cladosporium, the target gene sequence of Ochrasporium, the target gene sequence of Cladosporium, the target gene sequence of Vanadium, and the human RNase P gene sequence into the pUC 57 vector plasmid, respectively. After expansion culture, identification, and plasmid extraction, obtain the artificial synthetic plasmid of Helminthosporium, the artificial synthetic plasmid of Cladosporium, the artificial synthetic plasmid of Ochrasporium, the artificial synthetic plasmid of Cladosporium, the target gene sequence of Vanadium, and the internal reference artificial synthetic plasmid. The above artificial synthetic plasmids were quantified to 6×10 7 After 500 copies / mL, mix them in a volume ratio of 1:1:1:1:1:1 to make 60 μL of positive control, add 24 μL of lyophilization protectant, and lyophilize according to the lyophilization procedure in step (2) to obtain a lyophilized positive control.

[0083] Step 4: Assemble into a test kit

[0084] The kit comprises a freeze-dried PCR reaction system, a freeze-dried positive control and a freeze-dried negative control.

[0085] Step 5: Build the reaction program

[0086] The reaction procedure is shown in Table 3 below:

[0087] Table 3 Reaction procedures used in the present invention

[0088]

[0089] During the cycling step, fluorescence was detected at 60°C, and fluorescence signals of FAM, ROX, CY5, and VIC channels were collected.

[0090] Example 3 Kit linearity, sensitivity, repeatability and specificity detection

[0091] 1. Template preparation

[0092] The target gene sequences of Helminthosporium, Cladosporium, Ochrasporium, Cladosporium corytatum and Vansella were inserted into pUC 57 vector plasmids, respectively. After expansion culture, identification and plasmid extraction, artificial synthetic plasmids of Helminthosporium, Cladosporium, Ochrasporium, Cladosporium corytatum and Vansella were obtained. The above artificial synthetic plasmids were quantified to 1×10 7 After that, they were diluted to 1×10 with TE Buffer. 6 copies / mL, 1×10 5 copies / mL, 1×10 4copies / mL, 1×10 3 The quality control samples of Helminthosporium, Cladosporium, Ochrasporium, Cladosporium, Vancomyces, Epidermophyton, Trichophyton, Microsporum, Malassezia furfur, Black Trichoderma and White Trichoderma were extracted using the nucleic acid extraction or purification reagent of Qingdao Han Tang Biotechnology Co., Ltd. After extraction, 100 μL of each quality control nucleic acid solution was obtained.

[0093] Reconstitute the positive control lyophilized powder with 600 μL TE Buffer.

[0094] 2. Fluorescence PCR detection

[0095] The freeze-dried PCR reaction system of Example 2 was reconstituted with 25 μL of the positive control, 25 μL of the negative control, the above-mentioned artificially synthesized plasmids of each concentration, and the quality control nucleic acid solution. After oscillation mixing and brief centrifugation, the PCR reaction tube was placed on the Macrostone 96S fluorescent PCR instrument, the name of each sample was set, and the PCR amplification reaction was performed according to the reaction program in Table 3.

[0096] 3. Results Analysis

[0097] like Figure 1 As shown, the sensitivity of Helminthosporium was 200 copies / mL, with good linearity and correlation coefficient R 2 The repeatability is good and the CV is 0.4%.

[0098] like Figure 2 As shown, the sensitivity of Cladosporium was 200 copies / mL, with good linearity and correlation coefficient R 2 The repeatability is good and the CV is 0.8%.

[0099] like Figure 3 As shown in Figure 2, the sensitivity of Ochra was 200 copies / mL, with good linearity and a correlation coefficient of R 2 The repeatability is good and the CV is 0.7%.

[0100] like Figure 4 As shown, the sensitivity of Cladosporium was 200 copies / mL, with good linearity and correlation coefficient R 2 The repeatability is good and the CV is 1.0%.

[0101] like Figure 5 As shown in the figure, the sensitivity of Vanadium was 200 copies / mL, with good linearity and correlation coefficient R 2 The repeatability is good and the CV is 1.2%.

[0102] like Figure 6 As shown, except for Helminthosporium, Cladosporium, Ochrasporium, Cladosporium and Vancomyces, Epidermophyton, Trichophyton, Microsporum, Malassezia furfur, Black Trichoderma and White Trichoderma did not produce peaks, and the kit had good specificity.

[0103] Example 4 Kit Application

[0104] 1. Template preparation

[0105] Skin tissues infected with 3 cases of Helminthosporium spp., 2 cases of Cladosporium spp., 2 cases of Ochrasporium spp., 3 cases of Cladosporium spp., and 3 cases of Vancomyces spp. were collected clinically and extracted using nucleic acid extraction or purification reagents from Qingdao Han Tang Biotechnology Co., Ltd., to obtain 100 μL of nucleic acid solution.

[0106] 2. Fluorescence PCR detection

[0107] The freeze-dried PCR reaction system in Example 2 was reconstituted with 25 μL of the positive control, 25 μL of the negative control, and the nucleic acid solution obtained after nucleic acid extraction of the above 13 clinical samples. After oscillation mixing and brief centrifugation, the PCR reaction tube was placed on the Macrostone 96S fluorescent PCR instrument, the name of each sample was set, and the PCR amplification reaction was performed according to the reaction program in Table 3.

[0108] 3. Results Analysis

[0109] The test results of 13 clinical samples are as follows Figure 7 As shown in the figure, the negative control has no peak, and the positive control has peaks in all channels. All pathogen samples can be detected normally, and the peak position of the internal reference is stable, which is consistent with the previous diagnosis results.

[0110] It should be noted that the above-described embodiments of the present invention do not limit the protection scope of the present invention. Any technical solution obtained by equivalent replacement or equivalent transformation should be included in the protection scope of the present invention.

Claims

1. A primer-probe combination for detecting a pathogen of chromoblastomycosis, characterized in that: The primer-probe composition comprises an upstream primer designed for Helminthosporium, a downstream primer designed for Helminthosporium, a fluorescent probe designed for Helminthosporium, an upstream primer designed for Cladosporium, a downstream primer designed for Cladosporium, a fluorescent probe designed for Cladosporium, an upstream primer designed for Ochrasporium, a downstream primer designed for Ochrasporium, a fluorescent probe designed for Ochrasporium, an upstream primer designed for Cladosporium, a downstream primer designed for Cladosporium, a fluorescent probe designed for Cladosporium, an upstream primer designed for Vandelia, a downstream primer designed for Vandelia, a fluorescent probe designed for Vandelia, an upstream primer designed for an internal reference of human RNase P gene, a downstream primer designed for an internal reference of human RNase P gene, and a fluorescent probe designed for an internal reference of human RNase P gene; The upstream primer designed for Helminthosporium: the sequence is shown in SEQ ID NO.1; The downstream primer designed for Helminthosporium: the sequence is shown in SEQ ID NO.2; The fluorescent probe designed for Helminthosporium: the sequence is shown in SEQ ID NO.3; The upstream primer designed for Cladosporium: the sequence is shown in SEQ ID NO.4; The downstream primer designed for Cladosporium: the sequence is shown in SEQ ID NO.5; The fluorescent probe designed for Cladosporium: the sequence is shown in SEQ ID NO.6; The upstream primer designed for Ochraspora: the sequence is shown in SEQ ID NO.7; The downstream primer designed for Ochraspora: the sequence is shown in SEQ ID NO.8; The fluorescent probe designed for Ochraspora: the sequence is shown in SEQ ID NO.9; The upstream primer designed for Cladosporium: the sequence is shown in SEQ ID NO.10; The downstream primer designed for Cladosporium: the sequence is shown in SEQ ID NO.11; The fluorescent probe designed for Cladosporium: the sequence is shown in SEQ ID NO.12; The upstream primer designed for the genus Vancomyces: the sequence is shown in SEQ ID NO.13; The downstream primer designed for the genus Vancomyces: the sequence is shown in SEQ ID NO.14; The fluorescent probe designed for the genus Vancomyces: the sequence is shown in SEQ ID NO.15; The upstream primer designed for the human RNase P gene as an internal reference: the sequence is shown in SEQ ID NO.16; The downstream primer designed for the human RNase P gene as an internal reference: the sequence is shown in SEQ ID NO.17; The fluorescent probe designed as an internal reference for the human RNase P gene: the sequence is shown in SEQ ID NO.

18.

2. A kit for detecting the pathogen of chromoblastomycosis, characterized in that: It includes a freeze-dried PCR reaction system, a positive control and a negative control; the PCR reaction system includes a tube A PCR reaction system and a tube B PCR reaction system, and the tube A PCR reaction system and the tube B PCR reaction system are prepared according to 20 μL per person, and 8 μL of freeze-dried protective agent is added to each person, and the two PCR reaction tubes A and B are divided into two PCR reaction tubes; The primer combination provided in claim 1 is used to prepare reagents, and the PCR reaction system of tube A is prepared according to Table 1, and the PCR reaction system of tube B is prepared according to Table 2: Table 1 ； Table 2 ； In Table 1: the Bipolaris-F sequence is shown in SEQ ID NO.1, the Bipolaris-R sequence is shown in SEQ ID NO.2, the Bipolaris-P sequence is shown in SEQ ID NO.3, the Cladosporium-F sequence is shown in SEQ ID NO.4, the Cladosporium-R sequence is shown in SEQ ID NO.5, the Cladosporium-P sequence is shown in SEQ ID NO.6, the Ochronosis-F sequence is shown in SEQ ID NO.7, the Ochronosis-R sequence is shown in SEQ ID NO.8, the Ochronosis-P sequence is shown in SEQ ID NO.9, the IC-F1 sequence is shown in SEQ ID NO.16, the IC-R1 sequence is shown in SEQ ID NO.17, and the IC-P1 sequence is shown in SEQ ID NO.18; In Table 2: the Rhinocladiella-F sequence is shown as SEQ ID NO.10, the Rhinocladiella-R sequence is shown as SEQ ID NO.11, the Rhinocladiella-P sequence is shown as SEQ ID NO.12, the Wangiella-F sequence is shown as SEQ ID NO.13, the Wangiella-R sequence is shown as SEQ ID NO.14, the Wangiella-P sequence is shown as SEQ ID NO.15, the IC-F1 sequence is shown as SEQ ID NO.16, the IC-R1 sequence is shown as SEQ ID NO.17, and the IC-P1 sequence is shown as SEQ ID NO.

18.

3. A kit for detecting the pathogen of chromoblastomycosis according to claim 2, characterized in that: The two packaged PCR reaction systems were subjected to the following freeze-drying procedure: pre-freezing stage: pre-freezing at -45°C for 4 hours; main drying stage: evacuating the vacuum to 1-10Pa in 5 minutes, raising the shelf temperature to -40°C in 5 minutes; then keeping at -40°C for 10 hours; analytical drying stage: maintaining the vacuum at 1-10Pa, raising the shelf temperature to 30°C in 6.5 hours; then keeping at 30°C for 2 hours to obtain the freeze-dried PCR reaction system.

4. A kit for detecting the pathogen of chromoblastomycosis according to claim 2, characterized in that: The mass percentage concentrations of the components in the freeze-dried protective agent are: 5% polysucrose 400, 5% xylitol, 2% glutamic acid, and 1% vitamin E.

5. A kit for detecting the pathogen of chromoblastomycosis according to claim 2, characterized in that: The positive control is a plasmid mixture of an artificial synthetic plasmid of the genus Helminthosporium, an artificial synthetic plasmid of the genus Cladosporium, an artificial synthetic plasmid of the genus Ochrasporium, an artificial synthetic plasmid of the genus Cladosporium, an artificial synthetic plasmid of the genus Vanadium and an internal reference artificial synthetic plasmid, and the concentration ratio of the above artificial synthetic plasmids is 1:1:1:1:1:1:

1.

6. A kit for detecting the pathogen of chromoblastomycosis according to claim 2, characterized in that: The negative control is TE Buffer.

7. A kit for detecting the pathogen of chromoblastomycosis according to claim 5, characterized in that: The internal reference artificial synthetic plasmid is a plasmid into which a human RNase P gene sequence fragment is inserted.

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