Primer probe group, kit and detection method for simultaneously detecting five fungi

Abstract

The invention discloses a primer probe group, a kit and a detection method for simultaneously detecting five fungi. Specifically, in the primer and probe group, the nucleotide sequences of the primers comprise SEQ ID NO.7 to 8, SEQ ID NO.16 to 17, SEQ ID NO.25 to 26, SEQ ID NO.34 to 35 and SEQ ID NO.43 to 44, and the nucleotide sequences of the probes comprise SEQ ID NO.9, SEQ ID NO.18, SEQ ID NO.27, SEQ ID NO.36 and SEQ ID NO.45. The invention also discloses a kit for detecting the detection of the tumor cells. According to the invention, multiple detection in one tube is realized, and five invasive fungi, namely mucor, aspergillus, cryptococcus neoformans, pneumocystis yarrowii and talaromyces marneffei, are simultaneously detected in one tube of reagent by utilizing a PCR method.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a primer-probe set, a kit and a detection method for simultaneously detecting five fungi. Background Art

[0002] Fungal infections are relatively common in clinical practice. Currently, there are about more than 300 known pathogenic fungi, and most of them are opportunistic pathogens. According to the different parts of the human body invaded, pathogenic fungi can be divided into superficial fungi and deep fungi (invasive fungi). Invasive fungal diseases (IFI / IFDs) are deep fungi invading human tissues, blood and growing and multiplying in them, causing inflammatory reactions and dysfunction of tissues and organs. In recent years, due to the increase in the number of patients with malignant tumors, immunodeficiency diseases or organ transplants and the widespread use of broad-spectrum antibiotics, the incidence of IFDs has generally shown an upward trend, becoming a global public health problem.

[0003] The main invasive fungi include Cryptococcus, Aspergillus, Candida and Pneumocystis jirovecii, etc. Pathogens that were previously considered rare, such as Talaromyces marneffei and Mucor, are becoming more common. Due to the non-specific clinical manifestations of fungal infections, the current traditional fungal diagnostic methods, such as direct microscopy, culture and serological detection, etc., have low sensitivity and specificity and low detection rates, making it difficult to detect IFDs early and prone to missed diagnosis or misdiagnosis. Summary of the Invention

[0004] In order to solve the deficiencies existing in the prior art, the purpose of the present invention is to provide a method for simultaneously detecting multiple invasive fungi. The present invention realizes multi-detection in one tube, and uses the PCR method to simultaneously detect five invasive fungi, namely Mucor, Aspergillus spp., Cryptococcus neoformans, Pneumocystis jirovecii and Talaromyces marneffei in one tube of reagent.

[0005] The first aspect of the present invention provides a primer-probe set, wherein the nucleotide sequences of the primers include SEQ ID NO.7-8, SEQ ID NO.16-17, SEQ ID NO.25-26, SEQ ID NO.34-35 and SEQ ID NO.43-44, and the nucleotide sequences of the probes include SEQ ID NO.9, SEQ ID NO.18, SEQ ID NO.27, SEQ ID NO.36 and SEQ ID NO.45.

[0006] In some embodiments, the primer-probe set further includes endogenous internal standard primers and an endogenous internal standard probe. The nucleotide sequences of the endogenous internal standard primers include SEQ ID NO.52 and SEQ ID NO.53, and the nucleotide sequence of the endogenous internal standard probe includes SEQ ID NO.54.

[0007] In some embodiments, the probe is modified with a fluorescent group and a quenching group.

[0008] In the present invention, the fluorescent group is selected from any one of CY5, CY5.5, ROX, FAM, ATTO425, and VIC and are different from each other.

[0009] In the present invention, the quenching group is selected from any one of BHQ3, BHQ2, BHQ1, and MGB.

[0010] In some embodiments, the probe with the nucleotide sequence shown in SEQ ID NO.9 is modified with a fluorescent group of CY5 and a quenching group of BHQ3, the probe with the nucleotide sequence shown in SEQ ID NO.18 is modified with a fluorescent group of ROX and a quenching group of BHQ2, the probe with the nucleotide sequence shown in SEQ ID NO.27 is modified with a fluorescent group of FAM and a quenching group of BHQ1, the probe with the nucleotide sequence shown in SEQ ID NO.36 is modified with a fluorescent group of ATTO425 and a quenching group of MGB, and the probe with the nucleotide sequence shown in SEQ ID NO.45 is modified with a fluorescent group of VIC and a quenching group of BHQ1.

[0011] The second aspect of the present invention provides a kit, which includes the primer-probe set described in the first aspect of the present invention.

[0012] In some embodiments, the kit further includes an amplification reaction solution and nuclease-free water.

[0013] In some embodiments, the final concentration of each primer is 0.1-0.2 μM, and the final concentration of each probe is 0.1-0.2 μM.

[0014] As a preferred embodiment, the final concentration of the primer with the nucleotide sequence shown in SEQ ID NO.7-8 is 0.1 μM, the final concentration of the probe with the nucleotide sequence shown in SEQ ID NO.9 is 0.1 μM, the final concentration of the primer with the nucleotide sequence shown in SEQ ID NO.16-17 is 0.15 μM, the final concentration of the probe with the nucleotide sequence shown in SEQ ID NO.18 is 0.15 μM, the final concentration of the primer with the nucleotide sequence shown in SEQ ID NO.25-26 is 0.1 μM, the final concentration of the probe with the nucleotide sequence shown in SEQ ID NO.27 is 0.1 μM, the final concentration of the primer with the nucleotide sequence shown in SEQ ID NO.34-35 is 0.1 μM, the final concentration of the probe with the nucleotide sequence shown in SEQ ID NO.36 is 0.2 μM, the final concentration of the primer with the nucleotide sequence shown in SEQ ID NO.43-44 is 0.2 μM, and the final concentration of the probe with the nucleotide sequence shown in SEQ ID NO.45 is 0.1 μM.

[0015] The third aspect of the present invention provides the use of the primer-probe set described in the first aspect of the present invention or the kit described in the second aspect of the present invention in the preparation of a product for simultaneously detecting five invasive fungi, wherein the five invasive fungi include Mucor, Aspergillus, Cryptococcus neoformans, Pneumocystis jirovecii, and Talaromyces marneffei.

[0016] In some embodiments, the product includes a diagnostic reagent, a diagnostic device, or a diagnostic system.

[0017] The fourth aspect of the present invention provides a method for simultaneously detecting five invasive fungi for non-diagnostic purposes, the five invasive fungi include Mucor, Aspergillus, Cryptococcus neoformans, Pneumocystis jirovecii, and Talaromyces marneffei, wherein the method includes:

[0018] Extracting the DNA of the sample to be tested;

[0019] Performing a PCR reaction using the primer-probe set described in the first aspect of the present invention or the kit described in the second aspect of the present invention to detect whether one or more of the five invasive fungi are contained in the sample to be tested.

[0020] In some embodiments, the sample to be tested includes, but is not limited to, sputum, blood, urine, cerebrospinal fluid, tissue samples, cell samples, amniotic fluid, or feces.

[0021] In some embodiments, the PCR is a fluorescence quantitative PCR.

[0022] In some embodiments, the final concentration of each of the primers is 0.1 - 0.2 μM, and the final concentration of each of the probes is 0.1 - 0.2 μM.

[0023] As a preferred embodiment, the final concentration of the primer having the nucleotide sequence shown in SEQ ID NO.7 - 8 is 0.1 μM, the final concentration of the probe having the nucleotide sequence shown in SEQ ID NO.9 is 0.1 μM, the final concentration of the primer having the nucleotide sequence shown in SEQ ID NO.16 - 17 is 0.15 μM, the final concentration of the probe having the nucleotide sequence shown in SEQ ID NO.18 is 0.15 μM, the final concentration of the primer having the nucleotide sequence shown in SEQ ID NO.25 - 26 is 0.1 μM, the final concentration of the probe having the nucleotide sequence shown in SEQ ID NO.27 is 0.1 μM, the final concentration of the primer having the nucleotide sequence shown in SEQ ID NO.34 - 35 is 0.1 μM, the final concentration of the probe having the nucleotide sequence shown in SEQ ID NO.36 is 0.2 μM, the final concentration of the primer having the nucleotide sequence shown in SEQ ID NO.43 - 44 is 0.2 μM, and the final concentration of the probe having the nucleotide sequence shown in SEQ ID NO.45 is 0.1 μM.

[0024] As a preferred embodiment, the amplification conditions for the PCR reaction are: pre - treatment at 37°C for 2 min, pre - denaturation at 95°C for 2 min, denaturation at 95°C for 15 s, annealing and extension at 60°C for 30 s, and 40 cycles of denaturation, annealing and extension reactions are carried out.

[0025] Beneficial effects:

[0026] By virtue of the sensitive, specific and rapid characteristics of the molecular detection technology, the present invention can assist in the clinical diagnosis of early IFDs.

[0027] The present invention adopts the method of fluorescence PCR to simultaneously detect multiple fungi in the same tube, which has the advantages of good specificity and high sensitivity. Moreover, the addition of an endogenous internal standard can monitor the PCR process in real time, contributing to the rapid diagnosis of invasive fungal infections. At the same time, the commonly used detection method is to design primers for the internal transcribed spacer (ITS) region of ribosomal genes. However, in view of the disadvantages of high conservation and poor specificity of the ITS region in fungi, we have developed a primer - probe set, kit and method with strong specificity, high sensitivity, and capable of rapidly and accurately detecting five invasive fungi, namely Mucor, Aspergillus, Cryptococcus neoformans, Pneumocystis jirovecii, and Talaromyces marneffei.

[0028] In view of the above-mentioned identification and time requirements, the present invention has conducted a large number of sequence screening and alignments through NCBI (National Center for Biotechnology Information, hereinafter referred to as NCBI). Primers and probes are designed in the specific regions of five invasive fungi, namely Mucor, Aspergillus, Cryptococcus neoformans, Pneumocystis jirovecii, and Talaromyces marneffei, respectively, which can quickly detect the presence of Mucor, Aspergillus, Cryptococcus neoformans, Pneumocystis jirovecii, and Talaromyces marneffei. The lowest detection limit of the present invention can reach 1 CFU / PCR reaction, and the result can be obtained within 1 hour after sample treatment, significantly shortening the detection time. Description of the Drawings

[0029] Figure 1 For the feasibility verification results;

[0030] Figure 2 For the primer concentration screening results;

[0031] Figure 3 For the probe concentration screening;

[0032] Figure 4 For the cross-reaction results;

[0033] Figure 5 For the sensitivity detection results;

[0034] Figure 6 For the clinical sample detection results. Detailed Embodiments

[0035] Before further describing the specific implementation embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific implementation embodiments; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific implementation embodiments, rather than limiting the protection scope of the present invention.

[0036] When the embodiments give a numerical range, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials similar or equivalent to the methods, devices, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0037] Unless otherwise stated, the experimental methods, detection methods, and preparation methods not detailed in the present invention all adopt the conventional techniques in the art of this technology.

[0038] Example 1

[0039] 1. Specificity verification strains

[0040] For this PCR reaction system, the following strains were used for system specificity verification. The specificity verification strains are shown in Table 1.

[0041] Table 1 List of strains

[0042]

[0043] 2. Design and screening of PCR amplification primers and probes

[0044] The main objective of the present invention is to simultaneously identify Mucor, Aspergillus, Cryptococcus neoformans, Pneumocystis jirovecii, and Talaromyces marneffei. The target genes used are shown in detail in Table 2.

[0045] Table 2 Table of target genes

[0046]

[0047] Search for the full-length sequences of the genes listed in Table 1 in Genbank, and perform blast alignments on the gene sequences to be detected for Mucor, Aspergillus, Cryptococcus neoformans, Pneumocystis jirovecii, and Talaromyces marneffei respectively. Through alignment analysis, obtain the conserved and specific sequence segments of the target genes, and input the conserved and specific sequence segments of each gene into the design software respectively to design 3 pairs of primers and probes for detection targets. During the design process, not only the co-amplification problem of primers and probes for different target genes in one reaction system needs to be considered, that is, evaluate the Tm value, the difference in Tm values of the corresponding probes for the targets, the GC content, and avoid the occurrence of hairpin structures and dimers, etc., but also ensure that the alternative primer and probe segments can comprehensively cover Mucor, Aspergillus, Cryptococcus neoformans, Pneumocystis jirovecii, and Talaromyces marneffei. The primer and probe sequence table is shown in detail in Table 3.

[0048] Table 3 Primer and probe sequence table

[0049]

[0050]

[0051]

[0052] In this experiment, three sets of primer-probes were designed for each detection target (the labeled fluorescent quenching groups are not limited to those identified in the table), and a pair of primer-probes with good detection performance was selected for real-time fluorescence quantitative PCR detection. The primer and probe sequences are shown as No. 1 (screening for Mucor); No. 4 (screening for Aspergillus); No. 7 (screening for Cryptococcus neoformans); No. 10 (screening for Pneumocystis jirovecii); No. 13 (screening for Talaromyces marneffei).

[0053] To ensure quality control, an endogenous internal standard primer-probe was added to the reaction system, which can monitor the whole process from sampling, sample processing to amplification, avoiding false negative results. The primer and probe sequences are shown as No. 16 (screening for endogenous internal standard).

[0054] 3. Feasibility verification experiment of PCR amplification system

[0055] The five sets of primer-probes (No. 1, No. 4, No. 7, No. 10, No. 13) and one set of endogenous internal standard primer-probe (No. 16) screened were combined into the same system for feasibility verification, and the results are as Figure 1 shown. The results showed that the reaction system could detect 5 targets and the endogenous internal standard simultaneously.

[0056] 4. Confirmation of PCR amplification system and reaction conditions

[0057] 4.1 Amplification system

[0058] The primer usage concentrations of the five sets of primer-probes (No. 1, No. 4, No. 7, No. 10, No. 13) and one set of endogenous internal standard primer-probe (No. 16) were screened, and the results are as Figure 2 shown. The standard for primer screening was: small Ct value and an S-shaped curve. When there was no obvious difference in amplification CT and curve shape, the one with a smaller concentration was selected as the optimal concentration. Based on this, the optimal concentrations of the primers involved in this patent were screened out (as Figure 2 shown).

[0059] Using the determined primer concentrations, the probe usage concentrations of the five sets of primer-probes (No. 1, No. 4, No. 7, No. 10, No. 13) and one set of endogenous internal standard primer-probe (No. 16) were screened, and the results are as Figure 3 shown. The standard for probe screening was: small Ct value and an S-shaped curve. When there was no obvious difference in amplification CT and curve shape, the one with a smaller concentration was selected as the optimal concentration. Based on this, the optimal concentrations of the probes involved in this patent were screened out (as Figure 3 shown).

[0060] Based on the Ct values of the amplification curves and fluorescence intensity, the PCR amplification reaction system was finally determined as shown in Table 4.

[0061] Table 4 Amplification reaction system

[0062]

[0063]

[0064] 4.2 Amplification conditions

[0065] Through a large number of experimental tests, conditions such as annealing time, annealing temperature, and number of cycles were optimized, and finally the PCR amplification conditions were determined as shown in Table 5.

[0066] Table 5 Amplification conditions

[0067]

[0068] 5. Cross-reaction

[0069] Microorganisms that are closely related to the detection target species, have the same living environment, are likely to cause the same or similar clinical symptoms, and colonizing bacteria near the infection site were selected, including Enterococcus faecium, Streptococcus pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus epidermidis, Acinetobacter baumannii, Klebsiella pneumoniae, Candida tropicalis, Candida krusei, Streptococcus mitis, Enterococcus faecalis, Streptococcus pyogenes, Haemophilus influenzae, Candida glabrata, Staphylococcus aureus, Proteus mirabilis, Streptococcus agalactiae, Listeria monocytogenes, Enterobacter cloacae, Pseudomonas aeruginosa, Candida parapsilosis, Neisseria meningitidis, Micrococcus luteus, Rhodococcus equi, Listeria grayi, Acinetobacter junii, Haemophilus parainfluenzae, Neisseria sicca, Pseudomonas fluorescens, Aeromonas hydrophila, Candida lusitaniae, Legionella pneumophila as specific evaluation samples, and bacterial suspensions with a concentration greater than 10 5 CFU / mL were selected for cross-reaction verification, and the test results are as Figure 4 shown. The results showed that only the endogenous internal standard had amplification, and other samples had no amplification. Therefore, this reaction system has good specificity and can effectively distinguish target and non-target microbial nucleic acids.

[0070] 6. Sensitivity detection

[0071] Nucleic acid gradients of a 1:1:1:1:1 mixture of Mucor, Aspergillus fumigatus (Aspergillus), Cryptococcus neoformans, Pneumocystis jirovecii, and Talaromyces marneffei with an initial concentration of 10 5 CFU / PCR were serially diluted to 10 3 CFU / PCR, 10 2 CFU / PCR, 10 1 CFU / PCR, 10 0 CFU / PCR and used as templates for sensitivity evaluation, with each gradient repeated 3 times. The established system was used for verification, and the results are shown inFigure 5 , from Figure 5 it can be seen that the reaction system of the present application can achieve the detection of samples with 10 3 CFU / PCR, 10 2 CFU / PCR, 10 1 CFU / PCR, 10 0 CFU / PCR, thus indicating that the sensitivity of the reaction system provided by the present invention is 1 CFU / PCR.

[0072] 7. Detection of the lowest detection limit

[0073] The mixed DNA of Mucor, Aspergillus fumigatus (Aspergillus), Cryptococcus neoformans, Pneumocystis jirovecii, and Talaromyces marneffei extracted was gradient-diluted according to the lowest detectable concentration determined in Technical Solution 6, and the DNA with 10 CFU / PCR, 1 CFU / PCR, and 0.1 CFU / PCR was used as the template. Each concentration gradient was tested 10 times in duplicate, and the test results are shown in Table 6 in detail. The evaluation criterion is: the detection rate is 100%. According to the test results in Table 6, the detection rates of the 10 CFU / PCR and 1 CFU / PCR templates are 100%, and the detection rate of the 0.1 CFU / PCR template is less than 100%.

[0074] Table 6 Lowest detection limit

[0075]

[0076]

[0077] Using the DNA reacted with 1 CFU / PCR as the template, 20 repeated tests were performed, and the test results are shown in Table 7 in detail. The evaluation criterion is: the detection rate is 100%, and the CV of the Ct value is less than 5%. According to the test results in Table 7, the lowest detection limit of the detection system of the present application is: 1 CFU / PCR reaction.

[0078] Table 7 Lowest detection limit 2

[0079]

[0080] 8. Detection of clinical samples

[0081] For each pathogen, 5 positive clinical sputum samples were collected and detected in the following manner.

[0082] 8.1 Processing of sputum samples

[0083] The collected sputum was added with 4% NaOH solution or 0.1% DTT solution in a ratio of 1 to 4 times, shaken vigorously for 5 min, and left standing at room temperature for about 30 min. After complete liquefaction, it was used for detection.

[0084] 8.2 Nucleic Acid Extraction and Detection

[0085] Nucleic acid extraction, purification and amplification are carried out through the supporting POCT device independently developed by Kunpeng.

[0086] Prepare the amplification reaction solution according to the optimal amplification system conditions determined in Technical Solution 4, add the template extracted in 8.2, and then carry out amplification according to the optimal amplification conditions determined in Technical Solution 4. Determine the positive and negative according to the PCR amplification curve. See Table 8 and Figure 6 .

[0087] Table 8 Detection Results of Clinical Samples

[0088]

[0089]

[0090] 9. Kit Storage Stability Test

[0091] The present invention provides a kit for detecting Mucor, Aspergillus, Cryptococcus neoformans, Pneumocystis jirovecii and Talaromyces marneffei. The components of the kit include an amplification reaction solution (PCR amplification premix, containing taq enzyme, dNTP, magnesium ions, potassium ions, etc. required for amplification), a primer-probe mixture (the concentrations of the primers and probes in the mixture are the concentrations in the finally determined PCR amplification reaction system in Step 4), nuclease-free water, positive control, etc.

[0092] Take the nucleic acids of Mucor, Aspergillus fumigatus (Aspergillus), Cryptococcus neoformans, Pneumocystis jirovecii and Talaromyces marneffei mixed in equal proportions at 10 CFU / μL as the template. Place the assembled kit according to the storage conditions (-20°C), and take the kits at 0, 10, 15, 30, 60, 90, 120, 150, 180 and 360 days respectively for the storage period test. The detection results of the storage period are shown in Table 9.

[0093] Table 9 Results of Storage Period Test

[0094]

[0095] As can be seen from Table 9, when the kit is stored under the specified conditions, it is detected as positive during different storage periods, and the results show that the storage period of the kit is at least 1 year.

[0096] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A primer-probe set, wherein, The nucleotide sequences of the primers include SEQ ID NO.7-8, SEQ ID NO.16-17, SEQ ID NO.25-26, SEQ ID NO.34-35 and SEQ ID NO.43-44, and the nucleotide sequences of the probes include SEQ ID NO.9, SEQ ID NO.18, SEQ ID NO.27, SEQ ID NO.36 and SEQ ID NO.

45.

2. The primer-probe set according to claim 1, wherein, The primer-probe set further includes endogenous internal standard primers and endogenous internal standard probes. The nucleotide sequences of the endogenous internal standard primers include SEQ ID NO.52 and SEQ ID NO.53, and the nucleotide sequence of the endogenous internal standard probe includes SEQ ID NO.

54.

3. The primer-probe set according to claim 1 or 2, wherein, The probes are modified with a fluorescent group and a quenching group; The fluorescent group is selected from any one of CY5, CY5.5, ROX, FAM, ATTO425 and VIC and are different from each other; The quenching group is selected from any one of BHQ3, BHQ2, BHQ1 and MGB.

4. A kit, which includes the primer-probe set according to any one of claims 1-3.

5. The kit according to claim 4, which further includes an amplification reaction solution and nuclease-free water.

6. The kit according to claim 4 or 5, wherein, The final concentration of each primer is 0.1-0.2 μM, and the final concentration of each probe is 0.1-0.2 μM; Preferably, the final concentration of the primer with the nucleotide sequence shown in SEQ ID NO.7-8 is 0.1 μM, the final concentration of the probe with the nucleotide sequence shown in SEQ ID NO.9 is 0.1 μM, the final concentration of the primer with the nucleotide sequence shown in SEQ ID NO.16-17 is 0.15 μM, the final concentration of the probe with the nucleotide sequence shown in SEQ ID NO.18 is 0.15 μM, the final concentration of the primer with the nucleotide sequence shown in SEQ ID NO.25-26 is 0.1 μM, the final concentration of the probe with the nucleotide sequence shown in SEQ ID NO.27 is 0.1 μM, the final concentration of the primer with the nucleotide sequence shown in SEQ ID NO.34-35 is 0.1 μM, the final concentration of the probe with the nucleotide sequence shown in SEQ ID NO.36 is 0.2 μM, the final concentration of the primer with the nucleotide sequence shown in SEQ ID NO.43-44 is 0.2 μM, and the final concentration of the probe with the nucleotide sequence shown in SEQ ID NO.45 is 0.1 μM.

7. Use of the primer-probe set according to any one of claims 1-3 or the kit according to any one of claims 4-6 in a product for simultaneously detecting five invasive fungi, wherein, The five invasive fungi include Mucor, Aspergillus, Cryptococcus neoformans, Pneumocystis jirovecii and Talaromyces marneffei.

8. The application according to claim 7, wherein The product includes a diagnostic reagent, a diagnostic device or a diagnostic system.

9. A method for simultaneously detecting five invasive fungi for non-diagnostic purposes, the five invasive fungi including Mucor, Aspergillus, Cryptococcus neoformans, Pneumocystis jirovecii, and Talaromyces marneffei, wherein, The method includes: extracting the DNA of the sample to be tested; performing a PCR reaction using the primer-probe set according to claims 1-3 or the kit according to claims 4-6 to detect whether one or more of the five invasive fungi are contained in the sample to be tested.

10. The method according to claim 9, wherein, The sample to be tested includes any one or more of sputum, blood, urine, cerebrospinal fluid, tissue sample, cell sample, amniotic fluid or feces.

Images