Respiratory system fungal infection detection system, kit and preparation method thereof

Abstract

The invention provides a respiratory system fungal infection detection system, a kit and a preparation method thereof, and belongs to the technical field of biological detection. The primer probe composition used by the respiratory system fungal infection detection system comprises upstream and downstream primers and a fluorescent probe sequence designed aiming at paracoccus braziliensis, upstream and downstream primers and a fluorescent probe sequence designed aiming at histoplasma capsulatus, and upstream and downstream primers and a fluorescent probe sequence designed aiming at troptosporidium. The kit comprises upstream and downstream primers and fluorescent probe sequences designed for budgettes dermatitis, upstream and downstream primers and fluorescent probe sequences designed for Sirpolyspora sessilifolia, upstream and downstream primers and fluorescent probe sequences designed for talaromyces marneffei, upstream and downstream primers and fluorescent probe sequences designed for internal reference designed for ribonuclease P, upstream and downstream primers and fluorescent probe sequences designed for ribonuclease P, upstream and downstream primers and fluorescent probe sequences designed for ribonuclease P, and upstream and downstream primers and fluorescent probe sequences designed for ribonuclease P, the kit can be used for effectively detecting the fungi of the respiratory system, and ensures high accuracy, specificity and sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a respiratory fungal infection detection system, a kit and a preparation method thereof. Background Art

[0002] Fungi are eukaryotic organisms with spores and no chloroplasts. They are widely found in nature. Most fungi are beneficial to humans, but a few can cause serious diseases when they infect humans. The pathological changes and pathophysiological processes in which fungi invade human tissues and blood and grow and reproduce in them, leading to tissue damage, organ dysfunction and inflammatory response, are called invasive fungal disease (IFD), also known as invasive fungal infection. In recent years, due to the increase in the number of malignant tumors, immunodeficiency, transplant patients, long-term use of broad-spectrum antibiotics, prolonged indwelling catheters, etc., the incidence of invasive fungal diseases has been increasing year by year, mainly occurring in the central nervous system, digestive system, respiratory system, etc.; among them, the fungi most likely to infect the respiratory system are Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, Talaromyces marneffei, etc.

[0003] Due to the insidious onset and atypical clinical symptoms of invasive fungal diseases, early diagnosis is difficult. In addition, the emergence of new pathogens and multidrug-resistant strains has increased the difficulty of diagnosis and treatment. Early diagnosis, rational use of drugs, and preemptive treatment are the key to treating diseases and saving lives. Therefore, achieving early rapid multi-target diagnosis is conducive to guiding rational use of drugs and has great significance for improving patient survival rates.

[0004] At present, most of the methods for clinical pathogen detection in China are microscopic examination, culture, histopathology, and serological examination. These methods are time-consuming, have low accuracy, and false negatives cannot be ruled out. The 2019 guidelines included nucleic acid testing for the first time in the diagnosis criteria for IFD. Compared with conventional detection methods, nucleic acid testing has higher sensitivity and strong specificity, and can perform rapid multi-target diagnosis. Currently, there are also nucleic acid detection methods, such as: Chinese invention patent application CN202410266869.4 discloses a primer and probe for detecting invasive fungi. The patent is used for the detection and identification of Aspergillus, Mucorales, Cryptococcus neoformans, Pneumocystis jiroveci and Talaromyces marneffei. However, the patent has a long reaction time and low sensitivity of 1.5×10 2 The detection sample range is small and only applicable to sputum and bronchoalveolar lavage fluid.

[0005] Chinese invention patent application CN202311005500.X discloses a composition for joint detection of fungi and a kit and application containing the same, but the patent is used for the detection of capsular histoplasma, Scedosporium, Mucor, and Aspergillus, and the detection sensitivity of the patent is low, which is 500 copies / mL.

[0006] Chinese invention patent application CN202311047089.2 discloses a composition for detecting invasive fungi, but the patent is used for the detection of Fusarium, Histoplasma capsulatum, Mucorales fungi, and Cryptococcus, and the detection sensitivity of the patent is low, which is 500 copies / mL. Summary of the invention

[0007] In view of the above-mentioned technical defects, the present invention provides a respiratory fungal infection detection system, a kit and a preparation method thereof, which are used to detect six fungal pathogens, namely, Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium and Talaromyces marneffei. The kit can effectively detect Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium and Talaromyces marneffei, while ensuring high accuracy, specificity and sensitivity, and can complete the detection and obtain results in a short time, so that patients can be diagnosed and treated as early as possible.

[0008] The technical solution adopted by the present invention is: The gene sequences of Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei were obtained from the NCBI GeneBank database, and the gene sequences were compared to design the following primers and probes: SEQ ID NO.1: an upstream primer designed for Paracoccidioides brasiliensis, the sequence of the upstream primer is 5'-tgcgccctctggtattcc-3'; referred to as Paracoccidioides brasiliensis-F; SEQ ID NO.2: A downstream primer designed for Paracoccidioides brasiliensis, the sequence of which is 5'-catttcgggcacgtccac-3'; referred to as Paracoccidioides brasiliensis-R; SEQ ID NO.3: A fluorescent probe designed for Paracoccidioides brasiliensis, the sequence of which is 5'-cctgtccgagcgtcatttcaaccct-3'; referred to as Paracoccidioides brasiliensis-P; the 5' end of the fluorescent probe is labeled with a fluorescent group 6-FAM, and the 3' end is labeled with a quenching group BHQ1; SEQ ID NO.4: an upstream primer designed for Histoplasma capsulatum, the sequence of the upstream primer is 5'-cgctttcaacattgtcagtgg-3'; referred to as Histoplasma capsulatum-F; SEQ ID NO.5: A downstream primer designed for Histoplasma capsulatum, the sequence of which is 5'-acaagaatatggtttggcgttac-3'; referred to as Histoplasma capsulatum-R; SEQ ID NO.6: A fluorescent probe designed for Histoplasma capsulatum, the sequence of which is 5'-tcttccttgtcagcgtcaatactaatgcga-3'; referred to as Histoplasma capsulatum-P; the 5' end of the fluorescent probe is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group BHQ2; SEQ ID NO.7: an upstream primer designed for Coccidioides immitis, the sequence of the upstream primer is 5'-gcccaggtatcatagatgcc-3'; referred to as Coccidioides immitis-F; SEQ ID NO.8: A downstream primer designed for Coccidioides immitis, the sequence of which is 5'-ctgaccagtagcaagactacc-3'; referred to as Coccidioides immitis-R; SEQ ID NO.9: A fluorescent probe designed for Coccidioides immitis, the sequence of which is 5'-tcatcgtcgccaagatcaggagcc-3'; referred to as Coccidioides immitis-P; the 5' end of the fluorescent probe is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group BHQ3; SEQ ID NO.10: an upstream primer designed for Blastomyces dermatitidis, the sequence of the upstream primer is 5'-catcagtatcacatgctgctg-3'; abbreviated as Blastomyces dermatitidis-F; SEQ ID NO.11: A downstream primer designed for Blastomyces dermatitidis, the sequence of which is 5'-ctatcatataaaacacctccaacac-3'; abbreviated as Blastomyces dermatitidis-R; SEQ ID NO.12: A fluorescent probe designed for Blastomyces dermatitidis, the sequence of which is 5'-tcctgaagaaacaaaaccatgagctaaccc-3'; referred to as Blastomyces dermatitidis-P; the 5' end of the fluorescent probe is labeled with a fluorescent group 6-FAM, and the 3' end is labeled with a quenching group BHQ1; SEQ ID NO.13: An upstream primer designed for Scedosporium, the sequence of which is 5'-cgaacctccgtttcctcag-3'; referred to as Scedosporium-F; SEQ ID NO.14: A downstream primer designed for Scedosporium, the sequence of which is 5'-ggacccaatgcgagcttg-3'; referred to as Scedosporium-R; SEQ ID NO.15: A fluorescent probe designed for Scedosporium, the sequence of which is 5'-agacttactacgcagaaggcaaccgc-3'; referred to as Scedosporium-P; the 5' end of the fluorescent probe is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group BHQ2; SEQ ID NO.16: An upstream primer designed for Talaromyces marneffei, the sequence of the upstream primer is 5'-tgtgtgttgggcgtggtc-3'; referred to as Talaromyces marneffei-F; SEQ ID NO.17: A downstream primer designed for Talaromyces marneffei, the sequence of which is 5'-tcaaccgtggtaaaactgtgg-3'; referred to as Talaromyces marneffei-R; SEQ ID NO.18: A fluorescent probe designed for Talaromyces marneffei, the sequence of which is 5'-tgaccaacctccgccgatccgt-3'; referred to as Talaromyces marneffei-P; the 5' end of the fluorescent probe is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group BHQ3; SEQ ID NO.19: An upstream primer designed as an internal reference for ribonuclease P, the sequence of the upstream primer is 5'-agatttggacctgcgagcc-3'; referred to as ribonuclease PF; SEQ ID NO.20: A downstream primer designed as an internal reference for ribonuclease P, the sequence of which is 5'-gagcggctccacaagt-3'; referred to as ribonuclease PR; SEQ ID NO.21: A fluorescent probe designed as an internal reference for ribonuclease P, the sequence of which is 5'-ttctgacctgaaggctctgcgcg-3'; referred to as ribonuclease PP; the 5' end of the fluorescent probe is labeled with a fluorescent group VIC, and the 3' end is labeled with a quenching group BHQ2; The present invention uses human housekeeping gene ribonuclease P as an internal reference and 6 pathogens to simultaneously perform nucleic acid extraction, PCR amplification and other processes, and can monitor the entire detection process to ensure the validity of the results.

[0009] The probes of the present invention are modified with FAM, ROX, CY5 and VIC fluorescent groups respectively, and are divided into A reaction tube and B reaction tube, which meet the requirements of multi-channel and high-throughput detection. The A reaction tube involves the detection of three pathogens, namely Paracoccidioides brasiliensis, Histoplasma capsulatum and Coccidioides immitis, and the B reaction tube involves the detection of three pathogens, namely Blastomyces dermatitidis, Scedosporium and Talaromyces marneffei.

[0010] The quality control reagents include positive quality control and negative quality control; The positive quality control is: the concentration is 7×10 6 The positive quality control was prepared by mixing 10 copies / mL of Paracoccidioides brasiliensis artificial synthetic plasmid, Histoplasma capsulatum artificial synthetic plasmid, Coccidioides immitis artificial synthetic plasmid, Blastomyces dermatitidis artificial synthetic plasmid, Scedosporium artificial synthetic plasmid, Talaromyces marneffei artificial synthetic plasmid and RNase P artificial synthetic plasmid in a volume ratio of 1:1:1:1:1:1:1; The negative quality control is TE Buffer.

[0011] A method for preparing a respiratory fungal infection detection kit comprises the following steps: Prepare respiratory fungal infection detection system, prepare quality control reagents, assemble into detection kits and construct reaction procedures; The respiratory fungal infection detection system includes a tube A PCR detection system and a tube B PCR detection system: The A-tube PCR detection system includes: 1 μL of GUeasy Taq DNA polymerase with a concentration of 5U / μL, 5 μL of 5X Taq Buffer, 0.5 μL of 10mM deoxyribonucleoside triphosphate dNTP, 0.5 μL of 10 μM Paracoccidioides brasiliensis-F, 0.5 μL of 10 μM Paracoccidioides brasiliensis-R, 0.5 μL of 10 μM Paracoccidioides brasiliensis-P, 0.5 μL of 10 μM Histoplasma capsulatum-F, 0.5 μL of 10 μM Histoplasma capsulatum-R, 0.5 μL of 10 μM Histoplasma capsulatum-P, 0.5 μL of 10 μM Coccidioides immitis-F, 0.5 μL of 10 μM Coccidioides immitis-R 0.5μL, 10μM Coccidioides immitis-P 0.5μL, 10μM RNase PF 0.5μL, 10μM RNase PR 0.5μL, 10μM RNase PP 0.5μL, 10μL of lyoprotectant, the reagents were freeze-dried using a vacuum freeze dryer, and the template was added in an amount of 50μL.

[0012] The PCR detection system for tube B includes: 1 μL of GUeasy Taq DNA polymerase with a concentration of 5 U / μL, 5 μL of 5X Taq Buffer, 0.5 μL of deoxyribonucleoside triphosphate dNTP with a concentration of 10 mM, 0.5 μL of Blastomyces dermatitidis-F with a concentration of 10 μM, 0.5 μL of Blastomyces dermatitidis-R with a concentration of 10 μM, 0.5 μL of Blastomyces dermatitidis-P with a concentration of 10 μM, 0.5 μL of Scedosporium-F with a concentration of 10 μM, 0.5 μL of Scedosporium-R with a concentration of 10 μM, 0.5 μL of Scedosporium-P with a concentration of 10 μM, 0.5 μL of Talaromyces marneffei-F with a concentration of 10 μM, 0.5 μL of Talaromyces marneffei-R with a concentration of 10 μM, 0.5 μL of Talaromyces marneffei-P with a concentration of 10 μM 0.5μL, 10μM RNase PF 0.5μL, 10μM RNase PR 0.5μL, 10μM RNase PP 0.5μL, 10μL lyoprotectant, freeze-dry the reagents using a vacuum freeze dryer, and 50μL of template.

[0013] The reaction procedure was: 95°C for 15 s, 95°C for 5 s, 60°C for 10 s, and 40 cycles.

[0014] The above-mentioned nucleic acid sample to be tested is a DNA sample to be tested.

[0015] The beneficial effects of the present invention are as follows: the present invention can effectively detect Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei, ensuring high accuracy, specificity, and sensitivity, while completing the detection and obtaining the results in a short time, so that patients can be diagnosed and treated as early as possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 This is the fluorescence quantitative PCR result of the primer combination for detecting Paracoccidioides brasiliensis.

[0018] Figure 2 The figure shows the results of fluorescent quantitative PCR using the primer combination to detect Histoplasma capsulatum.

[0019] Figure 3 This is a graph showing the results of fluorescent quantitative PCR using a primer combination to detect Coccidioides immitis.

[0020] Figure 4 This is a fluorescence quantitative PCR result diagram of the primer combination for detecting Blastomyces dermatitidis.

[0021] Figure 5 This is a graph showing the results of fluorescent quantitative PCR using a primer combination to detect Scedosporium.

[0022] Figure 6 This is the result of fluorescence quantitative PCR using the primer combination to detect Talaromyces marneffei.

[0023] Figure 7 This is a fluorescence quantitative PCR result diagram of the primer combination for detecting Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei.

[0024] Figure 8 This is a fluorescence quantitative PCR result diagram showing the sensitivity of the primer combination in detecting Paracoccidioides brasiliensis.

[0025] Fig. 9 The figure shows the fluorescence quantitative PCR result of the primer combination in detecting the sensitivity of Histoplasma capsulatum.

[0026] Fig.10 The fluorescence quantitative PCR result diagram shows the sensitivity of the primer combination in detecting Coccidioides immitis.

[0027] Fig.11 The fluorescence quantitative PCR result diagram shows the sensitivity of the primer combination in detecting Blastomyces dermatitidis.

[0028] Fig.12 This is a graph showing the fluorescence quantitative PCR results of the primer combination for detecting the sensitivity of Scedosporium.

[0029] Fig.13 This is a fluorescence quantitative PCR result diagram showing the sensitivity of the primer combination in detecting Talaromyces marneffei.

[0030] Fig.14 This is a graph showing the specific fluorescence quantitative PCR results of the primer combination for detecting Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei.

[0031] Fig.15 This is the result of fluorescence quantitative PCR of Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei in sputum samples.

[0032] Fig.16 This is the result of fluorescence quantitative PCR detection using different sample kits processed in the same way. DETAILED DESCRIPTION

[0033] 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.

[0034] Example 1 A primer-probe composition for a respiratory fungal infection detection system The gene sequences of Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei were obtained from the NCBI GeneBank database, and the gene sequences were compared to design the following primers and probes: SEQ ID NO.1: an upstream primer designed for Paracoccidioides brasiliensis, the sequence of the upstream primer is 5'-tgcgccctctggtattcc-3'; referred to as Paracoccidioides brasiliensis-F; SEQ ID NO.2: A downstream primer designed for Paracoccidioides brasiliensis, the sequence of which is 5'-catttcgggcacgtccac-3'; referred to as Paracoccidioides brasiliensis-R; SEQ ID NO.3: A fluorescent probe designed for Paracoccidioides brasiliensis, the sequence of which is 5'-cctgtccgagcgtcatttcaaccct-3'; referred to as Paracoccidioides brasiliensis-P; the 5' end of the fluorescent probe is labeled with a fluorescent group 6-FAM, and the 3' end is labeled with a quenching group BHQ1; SEQ ID NO.4: an upstream primer designed for Histoplasma capsulatum, the sequence of the upstream primer is 5'-cgctttcaacattgtcagtgg-3'; referred to as Histoplasma capsulatum-F; SEQ ID NO.5: A downstream primer designed for Histoplasma capsulatum, the sequence of which is 5'-acaagaatatggtttggcgttac-3'; referred to as Histoplasma capsulatum-R; SEQ ID NO.6: A fluorescent probe designed for Histoplasma capsulatum, the sequence of which is 5'-tcttccttgtcagcgtcaatactaatgcga-3'; referred to as Histoplasma capsulatum-P; the 5' end of the fluorescent probe is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group BHQ2; SEQ ID NO.7: an upstream primer designed for Coccidioides immitis, the sequence of the upstream primer is 5'-gcccaggtatcatagatgcc-3'; referred to as Coccidioides immitis-F; SEQ ID NO.8: A downstream primer designed for Coccidioides immitis, the sequence of which is 5'-ctgaccagtagcaagactacc-3'; referred to as Coccidioides immitis-R; SEQ ID NO.9: A fluorescent probe designed for Coccidioides immitis, the sequence of which is 5'-tcatcgtcgccaagatcaggagcc-3'; referred to as Coccidioides immitis-P; the 5' end of the fluorescent probe is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group BHQ3; SEQ ID NO.10: an upstream primer designed for Blastomyces dermatitidis, the sequence of the upstream primer is 5'-catcagtatcacatgctgctg-3'; abbreviated as Blastomyces dermatitidis-F; SEQ ID NO.11: A downstream primer designed for Blastomyces dermatitidis, the sequence of which is 5'-ctatcatataaaacacctccaacac-3'; abbreviated as Blastomyces dermatitidis-R; SEQ ID NO.12: A fluorescent probe designed for Blastomyces dermatitidis, the sequence of which is 5'-tcctgaagaaacaaaaccatgagctaaccc-3'; referred to as Blastomyces dermatitidis-P; the 5' end of the fluorescent probe is labeled with a fluorescent group 6-FAM, and the 3' end is labeled with a quenching group BHQ1; SEQ ID NO.13: An upstream primer designed for Scedosporium, the sequence of which is 5'-cgaacctccgtttcctcag-3'; referred to as Scedosporium-F; SEQ ID NO.14: A downstream primer designed for Scedosporium, the sequence of which is 5'-ggacccaatgcgagcttg-3'; referred to as Scedosporium-R; SEQ ID NO.15: A fluorescent probe designed for Scedosporium, the sequence of which is 5'-agacttactacgcagaaggcaaccgc-3'; referred to as Scedosporium-P; the 5' end of the fluorescent probe is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group BHQ2; SEQ ID NO.16: An upstream primer designed for Talaromyces marneffei, the sequence of the upstream primer is 5'-tgtgtgttgggcgtggtc-3'; referred to as Talaromyces marneffei-F; SEQ ID NO.17: A downstream primer designed for Talaromyces marneffei, the sequence of which is 5'-tcaaccgtggtaaaactgtgg-3'; referred to as Talaromyces marneffei-R; SEQ ID NO.18: A fluorescent probe designed for Talaromyces marneffei, the sequence of which is 5'-tgaccaacctccgccgatccgt-3'; referred to as Talaromyces marneffei-P; the 5' end of the fluorescent probe is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group BHQ3; SEQ ID NO.19: An upstream primer designed as an internal reference for ribonuclease P, the sequence of the upstream primer is 5'-agatttggacctgcgagcc-3'; referred to as ribonuclease PF; SEQ ID NO.20: A downstream primer designed as an internal reference for ribonuclease P, the sequence of which is 5'-gagcggctccacaagt-3'; referred to as ribonuclease PR; SEQ ID NO.21: A fluorescent probe designed as an internal reference for ribonuclease P, the sequence of which is 5'-ttctgacctgaaggctctgcgcg-3'; referred to as ribonuclease PP; the 5' end of the fluorescent probe is labeled with a fluorescent group VIC, and the 3' end is labeled with a quenching group BHQ2; The above primers and probe plasmids were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0035] Example 2 A respiratory fungal infection detection system and kit Step 1: Prepare respiratory fungal infection detection system The primer combination provided in Example 1 was used to prepare a respiratory fungal infection detection system, which included a tube A PCR detection system and a tube B PCR detection system. The prepared tube A PCR detection system and tube B PCR detection system were freeze-dried, respectively, and 50 μL of template was added.

[0036] The A-tube PCR detection system includes: 1 μL of GUeasy Taq DNA polymerase with a concentration of 5U / μL, 5 μL of 5X Taq Buffer, 0.5 μL of 10mM deoxyribonucleoside triphosphate dNTP, 0.5 μL of 10 μM Paracoccidioides brasiliensis-F, 0.5 μL of 10 μM Paracoccidioides brasiliensis-R, 0.5 μL of 10 μM Paracoccidioides brasiliensis-P, 0.5 μL of 10 μM Histoplasma capsulatum-F, 0.5 μL of 10 μM Histoplasma capsulatum-R, 0.5 μL of 10 μM Histoplasma capsulatum-P, 0.5 μL of 10 μM Coccidioides immitis-F, 0.5 μL of 10 μM Coccidioides immitis-R 0.5μL, 10μM Coccidioides immitis-P 0.5μL, 10μM RNase PF 0.5μL, 10μM RNase PR 0.5μL, 10μM RNase PP 0.5μL, 10μL of lyoprotectant, the reagents were freeze-dried using a vacuum freeze dryer, and the template was added in an amount of 50μL.

[0037] The PCR detection system for tube B includes: 1 μL of GUeasy Taq DNA polymerase with a concentration of 5 U / μL, 5 μL of 5X Taq Buffer, 0.5 μL of deoxyribonucleoside triphosphate dNTP with a concentration of 10 mM, 0.5 μL of Blastomyces dermatitidis-F with a concentration of 10 μM, 0.5 μL of Blastomyces dermatitidis-R with a concentration of 10 μM, 0.5 μL of Blastomyces dermatitidis-P with a concentration of 10 μM, 0.5 μL of Scedosporium-F with a concentration of 10 μM, 0.5 μL of Scedosporium-R with a concentration of 10 μM, 0.5 μL of Scedosporium-P with a concentration of 10 μM, 0.5 μL of Talaromyces marneffei-F with a concentration of 10 μM, 0.5 μL of Talaromyces marneffei-R with a concentration of 10 μM, 0.5 μL of Talaromyces marneffei-P with a concentration of 10 μM 0.5μL, 10μM RNase PF 0.5μL, 10μM RNase PR 0.5μL, 10μM RNase PP 0.5μL, 10μL lyoprotectant, freeze-dry the reagents using a vacuum freeze dryer, and 50μL of template.

[0038] GUeasy Taq DNA polymerase and Taq Buffer were purchased from General Biotechnology (Anhui) Co., Ltd., and deoxyribonucleoside triphosphate dNTPs were purchased from Beijing Solebao Technology Co., Ltd.

[0039] Step 2, preparing quality control reagents; The quality control reagents include positive quality control and negative quality control. The positive quality control is: the concentration is 7×10 6 The artificial synthetic plasmids of Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, Talaromyces marneffei and RNase P were mixed in a volume ratio of 1:1:1:1:1:1:1 to prepare the positive quality control. The concentrations of the artificial synthetic plasmids of Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, Talaromyces marneffei and RNase P in the positive quality control were all 1×10 6 copies / mL; The artificial synthetic plasmids of Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, Talaromyces marneffei and RNase P were all purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0040] The negative quality control is TE Buffer.

[0041] Step 3: Assemble into a test kit The kit includes a respiratory fungal infection detection system and quality control reagents.

[0042] Step 4: Build the reaction program The reaction procedure was: 95°C for 15 s, 95°C for 5 s, 60°C for 10 s, and 40 cycles.

[0043] Figure 1 This is the fluorescence quantitative PCR result of the primer combination for detecting Paracoccidioides brasiliensis.

[0044] Figure 2 The figure shows the results of fluorescent quantitative PCR using the primer combination to detect Histoplasma capsulatum.

[0045] Figure 3 This is a graph showing the results of fluorescent quantitative PCR using a primer combination to detect Coccidioides immitis.

[0046] Figure 4 This is a fluorescence quantitative PCR result diagram of the primer combination for detecting Blastomyces dermatitidis.

[0047] Figure 5 This is a graph showing the results of fluorescent quantitative PCR using a primer combination to detect Scedosporium.

[0048] Figure 6 This is the result of fluorescence quantitative PCR using the primer combination to detect Talaromyces marneffei.

[0049] Figure 7 The results of fluorescence quantitative PCR of primer combination for detecting Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei; Tube A shows the results of fluorescence quantitative PCR for Paracoccidioides brasiliensis, Histoplasma capsulatum, and Coccidioides immitis; Tube B shows the fluorescence quantitative PCR results of Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei.

[0050] Example 3 Kit sensitivity determination 1. Template preparation The artificial synthetic plasmids of Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei were quantified to 10 5 copies / mL, 10-fold gradient dilution to 10 4 copies / mL, 10 3copies / mL, 10 2 copies / mL, 10 1 copies / mL, 10 0 copies / mL.

[0051] 2. Fluorescence PCR detection 10 4 copies / mL, 10 3 copies / mL, 10 2 copies / mL, 10 1 copies / mL, 10 0 50 μL of copies / mL of Paracoccidioides brasiliensis artificial synthetic plasmid, capsular Histoplasma capsulatum artificial synthetic plasmid, Coccidioides immitis artificial synthetic plasmid, Blastomyces dermatitidis artificial synthetic plasmid, Scedosporium artificial synthetic plasmid, and Talaromyces marneffei artificial synthetic plasmid were added to the respiratory fungal infection detection system prepared in Example 2, and the reaction was carried out according to the reaction procedure of 95°C for 15s, 95°C for 5s, and 60°C for 10s, and 40 cycles, and the fluorescence signals of 6-FAM, ROX, CY5, and VIC channels were collected.

[0052] 3. Result analysis: Figure 8 This is a fluorescence quantitative PCR result diagram showing the sensitivity of the primer combination in detecting Paracoccidioides brasiliensis.

[0053] Fig. 9 The figure shows the fluorescence quantitative PCR result of the primer combination in detecting the sensitivity of Histoplasma capsulatum.

[0054] Fig.10 The fluorescence quantitative PCR result diagram shows the sensitivity of the primer combination in detecting Coccidioides immitis.

[0055] Fig.11 The fluorescence quantitative PCR result diagram shows the sensitivity of the primer combination in detecting Blastomyces dermatitidis.

[0056] Fig.12 This is a graph showing the fluorescence quantitative PCR results of the primer combination for detecting the sensitivity of Scedosporium.

[0057] Fig.13 This is a fluorescence quantitative PCR result diagram showing the sensitivity of the primer combination in detecting Talaromyces marneffei.

[0058] The results showed that 6 targets (Paracidis brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, Talaromyces marneffei) 10 1 The detection rate of copies / mL is 100%, 10 0No peak of copies / mL was observed, indicating that 10 1 copies / mL is the detection limit concentration, and the final detection limit of the primer combination of the kit is confirmed to be 1×10 1 copies / mL.

[0059] Example 4 Specificity detection experiment 1. Template preparation: To verify the specificity of the system and composition of the present invention, 200 μL of Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Legionella pneumophila, Bordetella pertussis, Staphylococcus aureus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Mycobacterium tuberculosis, nontuberculous mycobacteria, Acinetobacter baumannii, Acinetobacter johnii, Escherichia coli, Rhodococcus equi, Candida albicans, Candida tropicalis, Candida krusei, Pneumocystis jiroveci, Aspergillus nidulans, Cryptococcus neoformans, influenza A virus, influenza B virus, respiratory syncytial virus, new coronavirus, Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei were taken respectively. The company's nucleic acid extraction or purification reagent was used for nucleic acid extraction, and 100 μL of each of the nucleic acids of Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Legionella pneumophila, Bordetella pertussis, Staphylococcus aureus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Mycobacterium tuberculosis, nontuberculous mycobacteria, Acinetobacter baumannii, Acinetobacter johnsonii, Escherichia coli, Rhodococcus equi, Candida albicans, Candida tropicalis, Candida krusei, Pneumocystis jiroveci, Aspergillus nidulans, Cryptococcus neoformans, influenza A virus, influenza B virus, respiratory syncytial virus, new coronavirus, Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei were obtained for later use.

[0060] 2. PCR testing 50 μL of Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Legionella pneumophila, Bordetella pertussis, Staphylococcus aureus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Mycobacterium tuberculosis, nontuberculous mycobacteria, Acinetobacter baumannii, Acinetobacter johnii, Escherichia coli, Rhodococcus equi, Candida albicans, Candida tropicalis, Candida krusei, Pneumocystis, Aspergillus nidulans, Cryptococcus neoformans, influenza A virus, influenza B virus, respiratory syncytial virus, new coronavirus, Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei nucleic acid were added to the PCR reaction reagent prepared in Example 2, and the reaction was carried out according to the reaction procedure of 95° C. for 15 s, 95° C. for 5 s, 60° C. for 10 s, and 40 cycles, and the 6-FAM, ROX, CY5, and VIC channel fluorescence signals were collected.

[0061] 3. Result analysis: Fig.14 The results of the primer combination for detecting Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei are shown in Figure 1. Tube A shows the results of Paracoccidioides brasiliensis, Histoplasma capsulatum, and Coccidioides immitis; Tube B shows the results of Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei are shown in Figure 1.

[0062] The results showed that except for the target pathogens (Paracospora brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei), the other pathogens were not amplified, indicating that the composition provided by the present invention has no nonspecific amplification and has high specificity.

[0063] Example 5 Application of a respiratory fungal infection detection system and kit 1. Template preparation Nucleic acid was extracted from 7 simulated sputum samples containing fungi. The 7 samples were: Sputum sample 1: simulated sputum sample containing only nucleic acid of Paracoccidioides brasiliensis; Sputum sample 2: simulated sputum sample containing only nucleic acid of Histoplasma capsulatum; Sputum sample 3: simulated sputum sample containing only nucleic acid of Coccidioides immitis; Sputum sample 4: simulated sputum sample containing only nucleic acid of Blastomyces dermatitidis; Sputum sample 5: simulated sputum sample containing only nucleic acid of Scedosporium; Sputum sample 6: simulated sputum sample containing only nucleic acid of T. marneffei; Sputum sample 7: Simulated sputum sample containing nucleic acids of Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei Take 200 μL of the above-mentioned samples to be tested and the negative quality control and positive quality control of the kit respectively, and use the nucleic acid extraction or purification reagents produced by Qingdao Han Tang Biotechnology Co., Ltd. for extraction. After extraction, 100 μL of nucleic acid is obtained for each of the 7 samples.

[0064] 2. PCR reaction 50 μL of each of sputum samples 1-3 and negative and positive controls were added to the PCR detection system of tube A in Example 2 for reaction, 50 μL of each of sputum samples 4-6 and negative and positive controls were added to the PCR detection system of tube B in Example 2 for reaction, and 50 μL of each of sputum sample 7 and negative and positive controls were added to the PCR detection system of tube A and tube B in Example 2 for reaction, respectively. The reaction was carried out according to the reaction procedure of 95° C. for 15 s, 95° C. for 5 s, 60° C. for 10 s, and 40 cycles, and the fluorescence signals of 6-FAM, ROX, CY5, and VIC channels were collected.

[0065] 3. Result analysis: Fig.15 This is the result of fluorescence quantitative PCR of Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei in sputum samples.

[0066] The test results of 7 simulated sputum samples are as follows Fig.15 As shown, the results indicate that 6 samples containing single fungal nucleic acids and 1 sample containing 6 mixed fungal nucleic acids can be accurately detected, indicating that the present invention is capable of jointly detecting and distinguishing Paracoccidioides brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Scedosporium, and Talaromyces marneffei.

[0067] Example 6 A respiratory fungal infection detection system and test sample of the kit The detection system in the present invention can be applied to different types of samples, including but not limited to sputum, nasopharyngeal swabs, alveolar lavage fluid, pleural effusion, etc. Its flexibility and diversity make the detection system have a wide range of applications, and can be used in the fields of biomedical research, clinical diagnosis, etc. At the same time, the design of the detection system also takes into account the characteristics and processing methods of different samples, ensuring the accuracy and reliability of the test results. Therefore, the detection system in the present invention can be widely applied to various types of samples, providing users with a convenient and efficient detection solution.

[0068] Example 7 A respiratory fungal infection detection system and a method for processing test samples of a kit 1. Template preparation: The present invention provides a universal processing method for samples such as sputum, nasopharyngeal swab, alveolar lavage fluid, pleural effusion and the like. Specifically, a universal sample processing solution is prepared from 5mol / L guanidine perchlorate, 50mmol / L Tris-HCl, 20mmol / L cyclohexanetetraacetic acid (CYDTA), 0.5% sodium lauroyl sarcosinate, and 0.1mmol / L β-mercaptoethanol, 50μL of the above four negative samples are mixed with 150μL of the sample processing solution, and then added to the nucleic acid extraction or purification reagent produced by Qingdao Han Tang Biotechnology Co., Ltd. for nucleic acid extraction, and 100μL of nucleic acid of each of the four samples of sputum, nasopharyngeal swab, alveolar lavage fluid, and pleural effusion is obtained for standby use.

[0069] 2. PCR detection: 50 μL of each of the obtained nucleic acids was added to the respiratory fungal infection detection system of Example 2 for reaction, and the reaction was carried out according to the reaction procedure of 95°C for 15 s, 95°C for 5 s, and 60°C for 10 s, and 40 cycles, and the fluorescence signals of 6-FAM, ROX, CY5, and VIC channels were collected.

[0070] 3. Result analysis: Fig.16 This is the result of fluorescence quantitative PCR detection using different sample kits processed in the same way.

[0071] As shown in the figure, the four negative sample internal references can all generate peaks, indicating that the processing method proposed by the present invention is suitable for processing these four samples. The above four samples can be selected as the samples to be tested in the present invention.

[0072] 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 respiratory fungal infection detection system, characterized in that: The primer-probe composition used in the respiratory fungal infection detection system includes an upstream primer designed for Paracoccidioides brasiliensis, a downstream primer designed for Paracoccidioides brasiliensis, a fluorescent probe designed for Paracoccidioides brasiliensis, an upstream primer designed for Histoplasma capsulatum, a downstream primer designed for Histoplasma capsulatum, a fluorescent probe designed for Histoplasma capsulatum, an upstream primer designed for Coccidioides immitis, a downstream primer designed for Coccidioides immitis, a fluorescent probe designed for Coccidioides immitis, an upstream primer designed for Blastomyces dermatitidis a fluorescent probe designed for Blastomyces dermatitidis, an upstream primer designed for Scedosporium, a downstream primer designed for Scedosporium, a fluorescent probe designed for Scedosporium, an upstream primer designed for Talaromyces marneffei, a downstream primer designed for Talaromyces marneffei, a fluorescent probe designed for Talaromyces marneffei, an upstream primer designed for an internal reference of the pseudoribonuclease P gene, a downstream primer designed for an internal reference of ribonuclease P, and a fluorescent probe designed for an internal reference of the ribonuclease P gene; The upstream primer designed for Paracoccidioides brasiliensis: the sequence is shown in SEQ ID NO.1; The downstream primer designed for Paracoccidioides brasiliensis: the sequence is shown in SEQ ID NO.2; The fluorescent probe designed for Paracoccidioides brasiliensis: the sequence is shown in SEQ ID NO.3; The upstream primer designed for Histoplasma capsulatum: the sequence is shown in SEQ ID NO.4; The downstream primer designed for Histoplasma capsulatum: the sequence is shown in SEQ ID NO.5; The fluorescent probe designed for Histoplasma capsulatum: the sequence is shown in SEQ ID NO.6; The upstream primer designed for Coccidioides immitis: the sequence is shown in SEQ ID NO.7; The downstream primer designed for Coccidioides immitis: the sequence is shown in SEQ ID NO.8; The fluorescent probe designed for Coccidioides immitis: the sequence is shown in SEQ ID NO.9; The upstream primer designed for Blastomyces dermatitidis has a sequence as shown in SEQ ID NO.10; The downstream primer designed for Blastomyces dermatitidis: the sequence is shown in SEQ ID NO.11; The fluorescent probe designed for Blastomyces dermatitidis has a sequence as shown in SEQ ID NO.12; The upstream primer designed for Scedosporium: the sequence is shown in SEQ ID NO.13; The downstream primer designed for Scedosporium: the sequence is shown in SEQ ID NO.14; The fluorescent probe designed for Scedosporium: the sequence is shown in SEQ ID NO.15; The upstream primer designed for Talaromyces marneffei has a sequence as shown in SEQ ID NO.16; The downstream primer designed for Talaromyces marneffei has a sequence as shown in SEQ ID NO.17; The fluorescent probe designed for Talaromyces marneffei: the sequence is shown in SEQ ID NO.18; The upstream primer designed for the internal reference of ribonuclease P: the sequence is shown in SEQ ID NO.19; The downstream primer designed for the internal reference of ribonuclease P: the sequence is shown in SEQ ID NO.20; The fluorescent probe designed as an internal reference for ribonuclease P: the sequence is shown in SEQ ID NO.

21.

2. A respiratory fungal infection detection system according to claim 1, characterized in that: Including A tube PCR detection system and B tube PCR detection system: The A-tube PCR detection system includes: 1 μL of GUeasy Taq DNA polymerase with a concentration of 5 U / μL, 5 μL of 5X Taq Buffer, 0.5 μL of 10 mM deoxyribonucleoside triphosphate dNTP, 0.5 μL of 10 μM Brazilian Paracoccidioides-F, 0.5 μL of 10 μM Brazilian Paracoccidioides-R, 0.5 μL of 10 μM Brazilian Paracoccidioides-P, 0.5 μL of 10 μM Capsular Histoplasma-F, 0.5 μL of 10 μM Capsular Histoplasma-R, 0.5 μL of 10 μM Capsular Histoplasma-P, 0.5 μL of 10 μM Coccidioides immitis-F, 0.5 μL of 10 μM Coccidioides immitis-R 0.5 μL, 10 μM Coccidioides immitis-P 0.5 μL, 10 μM RNase PF 0.5 μL, 10 μM RNase PR 0.5 μL, 10 μM RNase PP 0.5 μL, 10 μL of lyoprotectant, freeze-dried using a vacuum freeze dryer, 50 μL of template added; The PCR detection system for tube B includes: 1 μL of GUeasy Taq DNA polymerase with a concentration of 5U / μL, 5 μL of 5X Taq Buffer, 0.5 μL of 10mM deoxyribonucleoside triphosphate dNTP, 0.5 μL of 10 μM Blastomyces dermatitidis-F, 0.5 μL of 10 μM Blastomyces dermatitidis-R, 0.5 μL of 10 μM Blastomyces dermatitidis-P, 0.5 μL of 10 μM Scedosporium-F, 0.5 μL of 10 μM Scedosporium-R, 0.5 μL of 10 μM Scedosporium-P, 0.5 μL of 10 μM Talaromyces marneffei-F, 0.5 μL of 10 μM Talaromyces marneffei-R, 0.5 μL of 10 μM Talaromyces marneffei-P 0.5μL, 10μM RNase PF 0.5μL, 10μM RNase PR 0.5μL, 10μM RNase PP 0.5μL, 10μL lyoprotectant, freeze-dry the reagents using a vacuum freeze dryer, and 50μL of template.

3. A respiratory fungal infection detection kit, characterized in that: It includes respiratory fungal infection detection system and quality control reagents, and the quality control reagents include positive quality control and negative quality control.

4. A respiratory fungal infection detection kit according to claim 3, characterized in that: The positive quality control is as follows: the artificial synthetic plasmid of Paracoccidioides brasiliensis, the artificial synthetic plasmid of Histoplasma capsulatum, the artificial synthetic plasmid of Coccidioides immitis, the artificial synthetic plasmid of Blastomyces dermatitidis, the artificial synthetic plasmid of Scedosporium, the artificial synthetic plasmid of Talaromyces marneffei and the artificial synthetic plasmid of RNase P, all with a concentration of 7×106 copies / mL, are mixed in a volume ratio of 1:1:1:1:1:1:1:1 to prepare the positive quality control.

5. A respiratory fungal infection detection kit according to claim 3, characterized in that: The negative quality control is TE Buffer.

6. A method for preparing a respiratory fungal infection detection kit, characterized in that: The following steps are involved: A respiratory fungal infection detection system and quality control reagents were prepared, the detection kit was assembled, and a reaction program was constructed. The reaction program was as follows: 95° C. for 15 s, 95° C. for 5 s, 60° C. for 10 s, and 40 cycles.

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