Primers and probes, reagents and their use, kits and methods of detection for invasive fungi

Abstract

The application provides a set of primers and probes for detecting invasive fungi, reagents and application thereof, a kit and a detection method, and relates to the technical field of biology.The nucleic acid sequence of the primer is shown as SEQ ID NO.1-2, 4-5, 7-11, 14-15, 17-18 and 20-21; and the nucleic acid sequence of the probe is shown as SEQ ID NO.3, 6, 12-13, 16, 19 and 22.The primers and probes have high specificity and sensitivity, and can be used for the detection and identification of Aspergillus, Mucorales, Cryptococcus neoformans, Pneumocystis jirovecii and Lichtheimia. The kit for detecting invasive fungi has high specificity and sensitivity, and wide fungal coverage, and can realize the detection of multiple invasive fungi simultaneously.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a set of primers and probes, reagents, their applications, kits and detection methods for detecting invasive fungi. Background Technology

[0002] Fungi are eukaryotes, widely distributed in nature. The vast majority of fungi are beneficial to humans, while a small number can cause serious illness. Invasive fungal diseases (IFDs) refer to infectious diseases in which fungi invade the human body, grow and multiply in tissues, organs, or blood, leading to inflammatory responses and tissue damage. In recent years, the incidence and mortality rates of IFDs have been increasing annually, mainly due to their atypical early clinical manifestations and difficulty in early diagnosis, making them highly susceptible to misdiagnosis or missed diagnosis. Common fungal infections of the lungs include Aspergillus, Cryptococcus neoformans, Mucor, Pneumocystis jirovecii, and Bassilago farfara.

[0003] Due to the nonspecific clinical manifestations, limited detection techniques, difficulties in early detection, challenges in choosing treatment drugs, and poor prognosis associated with fungal infections, misdiagnosis and missed diagnoses are common. Early diagnosis, rational drug use, and prompt treatment are crucial for treating the disease and saving lives. Therefore, achieving early, rapid, and multi-target diagnosis is of great significance in guiding rational drug use and improving patient survival rates.

[0004] Currently, most clinical methods for pathogen detection in China involve microscopic examination, culture, histopathology, and serological testing. These methods have low positive rates, cannot rule out false negatives, and cannot differentiate at the species level. The 2019 guidelines for the diagnosis of IFD (Infectious Disease Fever) included evidence for the first time the use of tissue nucleic acid testing. When fungal components are observed in histopathology, fungal DNA can be amplified using polymerase chain reaction (PCR) combined with DNA sequencing. For example, Chinese patent application number 202311005500.X discloses a multiplex PCR method for detecting *Histoplasma capsulatum*, *Aspergillus*, *Mucor*, and *Cetus*. Another example is Chinese patent application number 202311047089.2, which discloses a method for detecting *Mucorales*, *Cryptococcus*, *Fusarium*, and *Histoplasma capsulatum* using RT-PCR, with a sensitivity of 500 copies / ml.

[0005] However, commonly used PCR detection methods for Aspergillus species share more than 95% sequence similarity with Penicillium. Therefore, fluorescent PCR techniques designed using conventional sequences cannot distinguish Penicillium, posing a risk of false positives. Several Mucor genera within the Mucorales order are pathogenic, and conventional techniques cannot cover all of them, leading to a risk of false negatives.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide primers and probes for detecting invasive fungi, which can be used for the detection of general fungi, Aspergillus, Mucorales, Cryptococcus neoformans, Pneumocystis jirovecii, and Bassilago farfara, thereby solving the aforementioned problems.

[0008] A second objective of this invention is to provide a reagent for detecting invasive fungi.

[0009] A third objective of this invention is to provide the application of the above-mentioned primers and probes or agents in the preparation of products for detecting invasive fungi.

[0010] A fourth objective of this invention is to provide a kit for detecting invasive fungi.

[0011] The fifth objective of this invention is to provide a method for detecting invasive fungi for non-disease diagnosis and treatment purposes, comprising: amplifying a sample using the aforementioned kit.

[0012] To achieve the above objectives, the following technical solution is adopted:

[0013] In a first aspect, the present invention provides primers and probes for detecting invasive fungi, the nucleic acid sequences of the primers being shown in SEQ ID NO. 1-2, 4-5, 7-11, 14-15, 17-18 and 20-21;

[0014] The nucleic acid sequences of the probes are shown in SEQ ID NO.3, 6, 12-13, 16, 19 and 22.

[0015] As a further technical solution, a fluorescent reporter group is attached to the 5' end of the probe, and a quencher group is attached to the 3' end;

[0016] The fluorescent reporter groups include FAM, VIC, ROX, and CY5;

[0017] The quenching groups include MGB, BHQ1, BHQ2 and BHQ3.

[0018] As a further technical solution, the fluorescent reporter groups of the probes with nucleic acid sequences such as SEQ ID NO.3, 12, and 16 are different from each other;

[0019] The fluorescent reporter groups of the probes with nucleic acid sequences such as SEQ ID NO.3, 13 and 16 are all different;

[0020] The fluorescent reporter groups of the probes with nucleic acid sequences such as SEQ ID NO.6, 19 and 22 are different from each other.

[0021] As a further technical solution, the invasive fungi include at least one of Aspergillus, Mucorales, Cryptococcus neoformans, Pneumocystis jirovecii, and Bassilago farfara.

[0022] Secondly, the present invention provides a reagent for detecting invasive fungi, comprising the aforementioned primers and probes.

[0023] Thirdly, the present invention provides the application of the above-mentioned primers and probes or agents in the preparation of products for detecting invasive fungi.

[0024] Fourthly, the present invention provides a kit for detecting invasive fungi, comprising the aforementioned primers and probes or reagents.

[0025] As a further technical solution, the kit includes reaction tube A and reaction tube B;

[0026] The A reaction tube includes primers with nucleic acid sequences as shown in SEQ ID NO. 1-2, 7-11 and 14-15, and probes with nucleic acid sequences as shown in SEQ ID NO. 3, 12-13 and 16;

[0027] The B reaction tube includes primers with nucleic acid sequences as shown in SEQ ID NO.4-5, 17-18 and 20-21, and probes with nucleic acid sequences as shown in SEQ ID NO.6, 19 and 22;

[0028] Preferably, the A reaction tube and the B reaction tube further include an internal standard primer and an internal standard probe; the nucleic acid sequence of the internal standard primer is shown in SEQ ID NO. 23 and 24; the nucleic acid sequence of the internal standard probe is shown in SEQ ID NO. 25.

[0029] As a further technical solution, an amplification reaction solution is also included.

[0030] Fifthly, the present invention provides a method for detecting invasive fungi for purposes other than disease diagnosis and treatment, comprising: amplifying a sample using the aforementioned kit.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The primers and probes provided by this invention for detecting invasive fungi are highly specific and sensitive, and can be used for the detection and identification of general fungi, Aspergillus, Mucorales, Cryptococcus neoformans, Pneumocystis jirovecii, and Bassilago farfara.

[0033] The kit for detecting invasive fungi provided by this invention can achieve rapid, comprehensive, sensitive, and specific detection that cannot be accomplished by pathogen culture, morphology, and immunological detection, achieving the following detection effects:

[0034] (i) High multiplex detection capability: Detecting a single sample can achieve multiplex detection of six pathogens at once: Fungi, Aspergillus, Muc, Cryptococcus neoformans, Pneumocystis japonicus, and Basilella marneffei (TM).

[0035] (II) Excellent coverage: It can cover common fungi that cause respiratory infections, including Aspergillus, Mucorales, Cryptococcus neoformans, Pneumocystis jirovecii, and Bassilago farfara. Among them, Aspergillus includes Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus oryzae, Aspergillus nidus, Aspergillus pyrophyllus, and Aspergillus versicolor; Mucorales includes Mucor spp, Rhizomucorspp, Rhizopus spp, and Lichtheimia.

[0036] (III) High specificity: In addition to being able to effectively distinguish from common respiratory pathogens, it can also be effectively distinguished from Penicillium, which has a high sequence similarity, thus achieving accurate detection.

[0037] (iv) Limit of detection: 150 copies / mL, which enables effective amplification of low-concentration samples. Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 The comparison results of selected regions of mitochondrial genes in Penicillium and Aspergillus genera. Detailed Implementation

[0040] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0041] In a first aspect, the present invention provides primers and probes for detecting invasive fungi, the nucleic acid sequences of the primers being shown in SEQ ID NO. 1-2, 4-5, 7-11, 14-15, 17-18 and 20-21;

[0042] The nucleic acid sequences of the probes are shown in SEQ ID NO.3, 6, 12-13, 16, 19 and 22.

[0043] The primers and probes provided by this invention for detecting invasive fungi are highly specific and sensitive, and can be used for the detection and identification of general fungi, Aspergillus, Mucorales, Cryptococcus neoformans, Pneumocystis jirovecii, and Bassilago farfara.

[0044] In some alternative embodiments, the probe has a fluorescent reporter group attached to its 5' end and a quencher group attached to its 3' end;

[0045] The fluorescent reporter groups include, but are not limited to, FAM, VIC, ROX, and CY5;

[0046] The quenching groups include, but are not limited to, MGB, BHQ1, BHQ2 and BHQ3.

[0047] In some alternative implementations, the fluorescent reporter groups of the probes with nucleic acid sequences such as SEQ ID NO. 3, 12, and 16 are different from each other;

[0048] The fluorescent reporter groups of the probes with nucleic acid sequences such as SEQ ID NO.3, 13 and 16 are all different;

[0049] The fluorescent reporter groups of the probes with nucleic acid sequences such as SEQ ID NO.6, 19 and 22 are different from each other.

[0050] To avoid non-specific amplification of primers during detection, primers and probes can be divided into two groups, with each group of probes having a different fluorescent group, in order to achieve the detection of fungi.

[0051] In some alternative embodiments, the invasive fungus includes at least one of Aspergillus, Mucorales, Cryptococcus neoformans, Pneumocystis yew, and Bassilago farfara.

[0052] The genus *Aspergillus* includes *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus niger*, *Aspergillus terreus*, *Aspergillus oryzae*, *Aspergillus nidus*, *Aspergillus pyrophorus*, and *Aspergillus variegata*; the order *Mucor* includes *Mucor spp*, *Rhizomucor spp*, *Rhizopus spp*, and *Lichtheimia*.

[0053] Secondly, the present invention provides a reagent for detecting invasive fungi, comprising the aforementioned primers and probes.

[0054] This reagent includes the primers and probes provided by this invention, and therefore has all the beneficial effects of the inventive primers and probes.

[0055] In some preferred embodiments, the reagents further include a PCR amplification system, which is purchased from Novizan Biotechnology, catalog number QN113-01.

[0056] The inventors discovered that this amplification system can cover Aspergillus species (Aspergillus fumigatus culture, Aspergillus flavus culture, Aspergillus niger culture, Aspergillus terreus culture, Aspergillus oryzae culture, Aspergillus nidus culture, and Aspergillus variegata culture), and there is no overlap with Penicillium species.

[0057] Thirdly, the present invention provides the application of the above-mentioned primers and probes or agents in the preparation of products for detecting invasive fungi.

[0058] The primers and probes for detecting invasive fungi provided by this invention have high specificity and sensitivity, and can be used for the detection and identification of Aspergillus, Mucorales, Cryptococcus neoformans, Pneumocystis jirovecii, and Bassilago farfara. Therefore, they can be used in the preparation of products for detecting invasive fungi.

[0059] Fourthly, the present invention provides a kit for detecting invasive fungi, comprising the aforementioned primers and probes or reagents.

[0060] The kit for detecting invasive fungi provided by this invention has high specificity, high sensitivity, and broad fungal coverage, and can simultaneously achieve multiplex detection of six pathogens: Fungi, Aspergillus, Muc, Cryptococcus neoformans, Pneumocystis japonicus, and Bassilago farfara (TM).

[0061] In some alternative implementations, the kit includes reaction tube A and reaction tube B;

[0062] The A reaction tube includes primers with nucleic acid sequences as shown in SEQ ID NO. 1-2, 7-11 and 14-15, and probes with nucleic acid sequences as shown in SEQ ID NO. 3, 12-13 and 16;

[0063] The B reaction tube includes primers with nucleic acid sequences as shown in SEQ ID NO.4-5, 17-18 and 20-21, and probes with nucleic acid sequences as shown in SEQ ID NO.6, 19 and 22.

[0064] By separating the primers and probes into two reaction tubes, non-specific amplification of the primers can be avoided.

[0065] In some optional embodiments, the A reaction tube and the B reaction tube further include internal standard primers and internal standard probes;

[0066] The nucleic acid sequences of the internal standard primers are shown in SEQ ID NO. 23 and 24;

[0067] The nucleic acid sequence of the internal standard probe is shown in SEQ ID NO.25.

[0068] In some alternative implementations, an amplification reaction solution is also included.

[0069] Fifthly, the present invention provides a method for detecting invasive fungi for purposes other than disease diagnosis and treatment, comprising: amplifying a sample using the aforementioned kit.

[0070] For example, the detection method provided by this invention can detect invasive fungi in water, food, etc., and assess the safety of water and food.

[0071] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0072] Example 1: Primer Pair and Probe Design and Synthesis

[0073] Table 1 Primer and Probe Information

[0074]

[0075]

[0076] In Table 1, W represents degenerate base A / T; Y represents degenerate base T / C; R represents degenerate base A / G; FAM in the probe is 6-carboxyfluorescein, VIC is 2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein, ROX is 6-carboxy-X-rhodamine, CY5 is 5H-indocyanine; MGB represents minor groove binder (MGB) and non-fluorescent quencher (NFQ).

[0077] Example 2: Screening of Aspergillus-specific primers and PCR amplification system

[0078] The mitochondrial gene of Aspergillus was selected as the target gene for detection. After sequence alignment analysis, a gene segment was selected as the amplicon sequence. The selected region differs from that of Penicillium by only one base (e.g., Figure 1As shown in Table 2, the primer design is as follows: (8th base from the bottom, other segments are not shown). In order to effectively distinguish and ensure coverage of common Aspergillus species in Aspergillus, the following 4 sets of primer probe schemes were designed to verify specificity and coverage.

[0079] Table 2. Information on Aspergillus primers and probes.

[0080]

[0081] The polymerase used in fluorescent PCR amplification is Taq DNA polymerase, which has 5'→3' polymerase activity and 5'→3' exonuclease activity, but lacks 3'→5' exonuclease activity. Therefore, this enzyme has no correction function if certain base mismatches occur during the PCR reaction. However, Taq DNA polymerases from different manufacturers have varying sensitivities to base mismatches due to differences in raw materials, processes, and modification methods. To further improve the distinguishability between Aspergillus and Penicillium schemes, PCR amplification systems from five manufacturers (Systems 1-5) were screened and paired with the above four schemes to form 20 test combinations for specificity and coverage verification. Product information for Systems 1-5 is as follows:

[0082] System 1: Purchased from Yisheng Biotechnology, product number 11211ES03;

[0083] System 2: Purchased from Novizan Biotechnology, product number Q112-02;

[0084] System 3: Purchased from Novizan Biotechnology, product number QN113-01;

[0085] System 4: Purchased from Takara Bio, product number R520A;

[0086] System 5: Purchased from Baorui Biotechnology, product number PM201.

[0087] Evaluation samples: Aspergillus fumigatus culture, Aspergillus flavus culture, Aspergillus niger culture, Aspergillus terreus culture, Aspergillus oryzae culture, Aspergillus nidus culture, Aspergillus variegata culture, and Penicillium culture.

[0088] The results of the verification are summarized below:

[0089] combination Can it cover Aspergillus species? Does it overlap with Penicillium? System 1 + Solution 1 no no System 1 + Solution 2 no yes System 1 + Solution 3 yes yes System 1 + Solution 4 yes yes System 2 + Plan 1 no no System 2 + Solution 2 yes yes System 2 + Plan 3 yes yes System 2 + Plan 4 yes yes System 3+Solution 1 no no System 3 + Solution 2 no no System 3 + Solution 3 yes yes System 3 + Plan 4 yes no System 4+Scheme 1 no no System 4 + Solution 2 no no System 4 + Solution 3 no no System 4 + Solution 4 yes yes System 5+ Plan 1 no no System 5+ Plan 2 no no System 5+Solution 3 yes yes System 5+ Plan 4 yes yes

[0090] As shown in the table above, the combination of System 3 and Scheme 4 can cover the above-mentioned Aspergillus species and has no overlap with Penicillium species.

[0091] Example 3: Screening of primer and probe schemes for Mucorales

[0092] The conserved gene ITS1+5.8S+ITS2 in the Mucorales order was selected, and primer and probe sequences were designed and synthesized. The sequence combinations are shown in the table below. The coverage of the scheme was verified for Mucor spp, Rhizomucor spp, Rhizopus spp, and Lichtheimia in the Mucorales order.

[0093]

[0094] Evaluation samples: Mucor cultures, Rhizopus cultures, Rhizopus cultures, and Rhizopus cultures.

[0095] It has been verified that Scheme 1 covers the four genera of Mucorales, while Scheme 2 does not cover the Rhizopus genus of Mucorales.

[0096] Example 4: Construction of primers, probes, and reagent kits

[0097] The kit contains the following components: PCR amplification reaction solution (system 3); 10× primer-probe mixture; positive control; negative control (enzyme-free sterile water).

[0098] The 10× primer-probe mixture in the kit includes two reaction tubes, A and B. The selection and combination of pathogens in each reaction tube are shown in Table 3.

[0099] Table 3. Selection and combination of pathogens

[0100]

[0101]

[0102] The blank nonspecific amplification index caused by primer-probe bridging was verified for 4 combinations of the above A and B reaction tubes. The verification showed that there was no nonspecific amplification in the blank control of combination 4.

[0103] Tube A contains the primers and probes listed in SEQ ID NO. 1-3, 7-16, and 23-25 ​​of Table 1; tube B contains the primers and probes listed in SEQ ID NO. 4-6 and 17-25 of Table 1.

[0104] Example 5: Kit Operation and Result Interpretation

[0105] 1. Collection of clinical samples

[0106] Liquefied sputum or bronchoalveolar lavage fluid samples were centrifuged at 12,500 rpm for 5 minutes. The resulting precipitate was mixed with 200 μL of physiological saline and centrifuged again at 12,500 rpm for 5 minutes. This process was repeated twice. After centrifugation and washing, the supernatant was removed. The resulting precipitate was then mixed with 50 μL of lysis buffer and nucleic acid extraction buffer, vortexed, and centrifuged at 12,500 rpm for 10 minutes. The supernatant was used as a template for PCR amplification.

[0107] 2. Preparation of the reaction system

[0108] Prepare the reaction system as follows: Total volume 20 μL, specific configuration as follows: PCR amplification reaction solution (system 3) 10 μL; 10× primer probe mixture A or B 2 μL; DNA template 8 μL.

[0109] 3. Reaction amplification

[0110] Place the PCR tube into a real-time PCR instrument, select FAM, VIC, CY5, and ROX as reporter groups, and follow the reaction procedure as follows:

[0111] a: 50℃, 2min;

[0112] b: 95℃, 1 min;

[0113] c: 95℃, 5s

[0114] d: 55℃, 30s; cd cycle for 40 reactions, during which fluorescence is collected.

[0115] 4. Interpretation of test results:

[0116] If the negative control, positive control, and internal standard control (which can only be used for amplification of internal standard primers and probes) are valid, and an S-shaped curve is detected in each fluorescence channel, then the detection result is considered valid.

[0117] If the FAM fluorescence channel of tube A is positive, the sample is determined to contain fungal nucleic acid; if the FAM and VIC channels of tube A are positive, the sample is determined to contain Mucorales nucleic acid; if the FAM and CY5 fluorescence channels of tube A are positive, the sample is determined to contain Cryptococcus neoformans nucleic acid.

[0118] If the detection results of the FAM and FAM fluorescence channels in tube A and tube B are positive, the sample is determined to contain Aspergillus nucleic acid; if the detection results of the FAM and VIC channels in tube A and tube B are positive, the sample is determined to contain Pneumocystis jirovecii nucleic acid; if the detection results of the FAM and CY5 fluorescence channels in tube A and tube B are positive, the sample is determined to contain Bassilago farfara nucleic acid.

[0119] Example 6: Specificity test of the kit

[0120] Microorganisms with homology to the nucleic acid sequences of the target lower respiratory tract infection bacteria, which are likely to cause the same or similar clinical symptoms, and which are normally present or easily co-occur at the sampling site, such as Penicillium, Bordetella pertussis, non-toxic Mycobacterium tuberculosis, Candida glabrata, Neisseria meningitidis, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, human coronavirus, cytomegalovirus, measles virus, human metapneumovirus, rhinovirus, adenovirus, respiratory syncytial virus, influenza virus, and parainfluenza virus, are selected as specificity evaluation samples.

[0121] Using the kit provided in this disclosure, the above-mentioned specificity evaluation samples were tested according to the detection method in Example 5. Under the condition that the negative control, positive control and internal standard control were all valid, no non-specific fluorescent signals were observed in the target samples, indicating that the kit of this disclosure can effectively distinguish non-target pathogens and has good specificity.

[0122] Example 7: Limit of Detection Test of the Kit

[0123] Evaluation cultures: Quantitatively quantified Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus oryzae, Aspergillus nidus, Aspergillus versicolor, Cryptococcus neoformans, Pneumocystis jirovecii, Mucor, Rhizopus, Rhizopus, Strombyx mori, and Bassilago farfara were serially diluted to obtain a concentration of 1.5 × 10⁻⁶. 3 copies / mL, 1.5×10 2 Evaluation templates of copies / mL and 1.5×10 copies / mL.

[0124] The templates for evaluation at each concentration were tested according to the detection method in Example 5. Each concentration gradient was tested 20 times, and the average value was taken as the final test result, as shown in Table 4.

[0125] Table 4. Validation results of the minimum detection limit

[0126]

[0127] Note: "+" indicates a positive result, and n / 20 represents a detection rate of 20 repeated n detections.

[0128] As can be seen from the table above, the limit of detection for each pathogen in the kit of this invention reaches 1.5 × 10⁻⁶. 2 The detection sensitivity is relatively high, with copies / mL.

[0129] Comparative Example

[0130] 1. Primer and probe synthesis

[0131] Sequence synthesis was performed according to the primer and probe sequences shown in Table 5.

[0132] Table 5 Primer and Probe Table

[0133]

[0134]

[0135] 2. Specificity verification

[0136] Following the procedure in Example 6, the results showed that there was overlap between Aspergillus and Penicillium, indicating poor specificity.

[0137] 3. Coverage Verification

[0138] Evaluation samples: Mucor cultures, Rhizopus cultures, Rhizopus cultures, and Rhizopus cultures.

[0139] The results showed that the comparative scheme could not cover the genera Rhizopus and Rhizopus in the Mucorales order, and there were false negatives.

[0140] 4. Validation of the lowest detection limit

[0141] Following the procedure in Example 7, the results are shown in Table 6.

[0142] Table 6. Validation results of the minimum detection limit

[0143]

[0144]

[0145] Note: "+" indicates a positive result, and n / 20 represents a detection rate of 20 repeated n detections.

[0146] As shown in the table above, the limit of detection for each pathogen using this kit is 1.5 × 10⁻⁶. 3 The results were poorer than those in Example 7, with a volume of copies / mL.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kit for detecting invasive fungi, characterized in that, The kit includes reaction tube A and reaction tube B; The A reaction tube includes primers with nucleic acid sequences as shown in SEQ ID NO. 1-2, 7-11 and 14-15, and probes with nucleic acid sequences as shown in SEQ ID NO. 3, 12-13 and 16; The B reaction tube includes primers with nucleic acid sequences as shown in SEQ ID NO. 4-5, 17-18 and 20-21, and probes with nucleic acid sequences as shown in SEQ ID NO. 6, 19 and 22; It also includes a PCR amplification system, which was purchased from Novizan Biotechnology, catalog number QN113-01; The invasive fungus is at least one of the following: Aspergillus, Mucorales, Cryptococcus neoformans, Pneumocystis yew, and Bassilago farfara.

2. The reagent kit according to claim 1, characterized in that, The A reaction tube and B reaction tube further include an internal standard primer and an internal standard probe; the nucleic acid sequences of the internal standard primers are shown in SEQ ID NO. 23 and 24; the nucleic acid sequence of the internal standard probe is shown in SEQ ID NO.

25.

3. The reagent kit according to claim 1, characterized in that, The probe has a fluorescent reporter group attached to its 5' end and a quencher group attached to its 3' end. The fluorescent reporter groups include FAM, VIC, ROX, and CY5; The quenching groups include MGB, BHQ1, BHQ2 and BHQ3.

4. The reagent kit according to claim 3, characterized in that, The fluorescent reporter groups of the probes with nucleic acid sequences such as SEQ ID NO.3, 12, and 16 are all different. The fluorescent reporter groups of the probes with nucleic acid sequences such as SEQ ID NO.3, 13 and 16 are all different; The fluorescent reporter groups of the probes with nucleic acid sequences such as SEQ ID NO. 6, 19 and 22 are different from each other.

5. A method for detecting invasive fungi for non-disease diagnosis and treatment purposes, characterized in that, include: The sample was amplified using the kit described in any one of claims 1-4.

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