Product for detecting common pathogens of invasive fungal disease

Abstract

The invention discloses a product for detecting common pathogens of invasive fungal diseases, and belongs to the technical field of molecular biological detection. The sequences of the primers are as shown in SEQ ID NO.5-6, 8-9, 11-12, 14-15, 17-18, 20-21, 23-24, 26-27, 29-30, 32-33, 35-36, 38-39 and 41-42, and the sequences of the probes are as shown in SEQ ID NO.4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37 and 40. The kit provided by the invention can qualitatively detect 13 common invasive fungi in human alveolar lavage fluid, has the advantages of high specificity and high sensitivity, can still detect under the condition of low nucleic acid content, and compared with mNGS, the kit has the advantages that the detection cost is low, the overall medical cost can be reduced, the detection period is short, one-time detection can be completed only in 1.5-2 hours, and the detection cost is low. The device is suitable for most common instruments, is easy to operate and can be widely applied to medical institutions at all levels.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology detection, and in particular relates to a product for detecting common pathogens of invasive fungal diseases. Background Art

[0002] Invasive fungal disease (IFD) refers to an infectious disease in which fungi invade the human body, reproduce and grow in tissues, organs or blood, and cause inflammatory reactions and tissue damage. In recent years, with the increasing complexity of clinical diagnosis and treatment plans, the widespread use of various invasive procedures, broad-spectrum antibiotics, immunosuppressants, glucocorticoids and tumor chemotherapy drugs, the incidence and mortality of the disease have increased year by year, mainly because its early clinical manifestations are atypical, early diagnosis is difficult, and it is very easy to misdiagnose or miss the diagnosis. According to the survey report on the current status of diagnosis and treatment of fungal diseases in China issued by Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, it is learned that invasive fungal disease has become a common problem faced by many related clinical departments such as blood, infection, respiratory, and intensive care. According to statistics, about 300 million people suffer from serious fungal infections every year worldwide, of which the annual incidence of invasive fungal disease is about 27.2 cases per 100,000 cases, and it increases at a rate of 0.9% per year, with an average mortality rate of about 27.6%. For high-risk groups with immune deficiencies such as malignant tumors, blood diseases, respiratory diseases, AIDS and severe liver disease, the mortality rate can be as high as 39% to 100% once invasive fungal infection occurs.

[0003] According to statistics, the main pathogens of invasive fungal diseases are Candida, Aspergillus, Mucor, and Cryptococcus. On October 25, 2022, the World Health Organization (WHO) published its first "Priority Fungal Pathogen List" (FPPL), which lists 19 fungi that pose a public health risk. These pathogens are categorized into severe, high, and intermediate groups based on their priority. Over the past few decades, despite data gaps, estimates of the morbidity and mortality of fungal infections have gradually accumulated, which is of great significance for public health planning and combating antifungal resistance. Multiple studies have estimated the morbidity and mortality of various fungal diseases, covering a large portion of the global population. David W. Denning, a core technical expert at the University of Manchester, UK, and a member of the Global Fungal Infections Action Group, published a review article in the journal Lancet Infectious Diseases titled "Global Incidence and Mortality of Severe Fungal Disease." The article summarizes current data on the morbidity and mortality of life-threatening fungal diseases, making important transparent assumptions about the proportion of confirmed and undiagnosed cases. It also provides the first estimate of attributable mortality, excluding cases of severe fungal infections that ultimately lead to death due to underlying medical conditions. Clinically, rapid and accurate laboratory diagnostic methods are needed for the early diagnosis of invasive fungal infections to improve patient survival. Molecular biological methods, particularly polymerase chain reaction (PCR), have become important approaches for fungal pathogen detection, gaining widespread consensus among experts and incorporated into the diagnostic evidence for fungal infections. Compared with traditional methods (microscopy and culture), PCR offers advantages such as high sensitivity, strong specificity, and rapid detection.

[0004] In February 2022, the Fungal Diseases Professional Committee of the Chinese Medical Education Association and the National Clinical Research Center for Skin and Immunological Diseases formulated the "Expert Consensus on the Clinical Application of Laboratory Diagnostic Methods for Invasive Fungal Diseases". This consensus stated in the molecular biology method that the fluorescent quantitative PCR method has application prospects in the detection of various deep infection fungi (Aspergillus, Pneumocystis, Candida). Fluorescent quantitative PCR also has application prospects in the detection of other fungal pathogens that are not Aspergillus infections. Domestic research institutions and hospitals are actively carrying out research on the precise diagnosis of invasive fungal infections. For example, the research team of Jinan University has developed a new sensor that can detect biomarkers of fungal infection in a short time, significantly improving the detection efficiency. Youkang Company has launched a fungal detection kit (qPCR method), but its functions are not comprehensive, do not include nucleic acid extraction function, and its accuracy needs to be improved.

[0005] In March 2021, CID published a supplement to the 2019 EORTC / MSG IFD guidelines, in which molecular diagnostic technology (PCR) was included in the fourth article. (1) One of the criteria for fungal diagnosis: When fungi are found in formalin-fixed, paraffin-embedded tissues, PCR amplification of fungal DNA is performed and combined with DNA sequencing; (2) One of the criteria for yeast diagnosis: When yeast are found in formalin-fixed, paraffin-embedded tissues, PCR amplification of fungal DNA is performed and combined with DNA sequencing. In the EORTC / MSGERC consensus guidelines, the combination of Aspergillus PCR detection and antigen detection is considered the best application. Molecular point-of-care testing (POCT) products have been widely used in clinical diagnosis internationally, especially in the detection of respiratory pathogens. For example, Cepheid's GeneXpert platform in the United States has carried out more than 40 detection projects, including tuberculosis and rifampicin resistance. Polymerase chain reaction (PCR) technology is widely used in the detection of fungal nucleic acids to improve the diagnostic accuracy of fungal infections. For example, the FilmArray platform of bioMérieux in the United States is mainly used for in vitro qualitative detection of nucleic acids of various pathogens in cerebrospinal fluid samples. Summary of the Invention

[0006] The present invention provides a primer and probe for multiplex PCR detection of respiratory pathogens, comprising:

[0007] Primers of the nucleotide sequences shown in SEQ ID NOs. 5-6 and a probe of the nucleotide sequence shown in SEQ ID NO. 4 for detecting Candida parapsilosis;

[0008] Primers of the nucleotide sequences shown in SEQ ID NOs. 8-9 and a probe of the nucleotide sequence shown in SEQ ID NO. 7 for detecting Cryptococcus neoformans;

[0009] Primers of the nucleotide sequences shown in SEQ ID NOs. 11-12 and a probe of the nucleotide sequence shown in SEQ ID NO. 10 for detecting Aspergillus fumigatus;

[0010] Primers of the nucleotide sequences shown in SEQ ID NOs. 14-15 and a probe of the nucleotide sequence shown in SEQ ID NO. 13 for detecting Candida albicans;

[0011] Primers of the nucleotide sequences shown in SEQ ID NOs. 17-18 and a probe of the nucleotide sequence shown in SEQ ID NO. 16 for detecting Pseudomonas aeruginosa;

[0012] Primers of the nucleotide sequences shown in SEQ ID NOs. 20-21 and a probe of the nucleotide sequence shown in SEQ ID NO. 19 for detecting Rhizomucor pusillus;

[0013] Primers of the nucleotide sequences shown in SEQ ID NOs. 23-24 and a probe of the nucleotide sequence shown in SEQ ID NO. 22 for detecting Candida tropicalis;

[0014] Primers of the nucleotide sequences shown in SEQ ID NOs. 26-27 and a probe of the nucleotide sequence shown in SEQ ID NO. 25 for detecting Aspergillus flavus;

[0015] Primers of the nucleotide sequences shown in SEQ ID NOs. 29-30 and a probe of the nucleotide sequence shown in SEQ ID NO. 28 for detecting Aspergillus niger;

[0016] Primers of the nucleotide sequences shown in SEQ ID NOs. 32-33 and a probe of the nucleotide sequence shown in SEQ ID NO. 31 for detecting Candida glabrata;

[0017] Primers of the nucleotide sequences shown in SEQ ID NOs. 35-36 and a probe of the nucleotide sequence shown in SEQ ID NO. 34 for detecting Candida krusei;

[0018] Primers of the nucleotide sequences shown in SEQ ID NOs. 38-39 and a probe of the nucleotide sequence shown in SEQ ID NO. 37 for detecting Aspergillus terreus;

[0019] Primers of the nucleotide sequences shown in SEQ ID NO. 41-42 and a probe of the nucleotide sequence shown in SEQ ID NO. 40 for detecting Rhizopus stolonifer.

[0020] The present invention also provides the use of the primers and probes in preparing products for detecting respiratory pathogens.

[0021] In one embodiment of the present invention, the product is a reagent or a kit.

[0022] The present invention also provides a product for detecting respiratory pathogens, which contains the above primers and probes.

[0023] In one embodiment of the present invention, the product is a reagent or a kit.

[0024] In one embodiment of the present invention, the kit further includes a detection reagent and a nucleic acid extraction reagent.

[0025] In one embodiment of the present invention, the detection reagent includes sterile water and PCR Mix.

[0026] In one embodiment of the present invention, the product further contains primers and probes for detecting internal reference genes.

[0027] In one embodiment of the present invention, the primer sequences for detecting the internal reference gene are shown as SEQ ID NO.2-3, and the probe sequence is shown as SEQ ID NO.1.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The kit provided by the present invention adopts the principle of Taq enzyme nucleic acid amplification technology. The kit contains specific primers and specific fluorescent probes designed for the conserved regions of the bacterial nucleic acid to be detected, and is equipped with components such as PCR reaction solution. On a fluorescent quantitative PCR instrument, multiple real-time fluorescent quantitative PCR detection technology is applied to achieve rapid detection of nucleic acids of lower respiratory pathogens in the test sample through changes in fluorescent signals. The PCR detection system includes primers and probes for human endogenous housekeeping genes (internal standards). By detecting whether the human internal standard is normal, it monitors whether the sample has been added and whether there are PCR inhibitors in the test sample to avoid PCR false negatives.

[0030] Existing clinical products, such as the fungal detection kit (qPCR method) launched by Youkang Company and the fungal DNA detection kit (PCR-fluorescent probe method) launched by Zhengyang Company, do not include nucleic acid extraction functions and have few fluorescent channels. They are not comprehensive and can only detect whether the sample contains fungi, but cannot accurately and qualitatively detect the species and category of the fungi.

[0031] The kit provided by the present invention can qualitatively detect 13 common invasive fungi in human alveolar lavage fluid, including 13 invasive fungi such as Rhizopus, Rhizomucor, Rhizobacter, Aspergillus, Cryptococcus, Mucor and Candida. Different fungal species and strains can be distinguished by designing specific primers and probes, which is of great significance for clinical treatment decisions and the selection of antifungal drugs. The primers and probes provided by the present invention have the advantages of high specificity and high sensitivity, and can still be detected when the amount of nucleic acid is low. Compared with mNGS, the detection cost is affordable, which can reduce the overall medical cost, and the detection cycle is short, and a test can be completed in only 1.5h-2h. It is suitable for most common instrument models, easy to operate, and can be widely used in medical institutions at all levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1-1 、 1-2 It is the detection result of Candida albicans in Example 1.

[0033] Figure 1-3 、 1-4 These are the test results of Aspergillus flavus and Candida glabrata in Example 1, where ① represents Aspergillus flavus and ② represents Candida glabrata.

[0034] Figure 1-5 、 1-6The test results of Aspergillus niger in Example 1 are shown in FIG.

[0035] Figure 1-7 、 1-8 These are the detection results of Cryptococcus neoformans, Candida parapsilosis, and Aspergillus fumigatus in Example 1, where ① represents Cryptococcus neoformans, ② represents Candida parapsilosis, and ③ represents Aspergillus fumigatus.

[0036] Figure 1-9 、 1-10 These are the test results of Candida tropicalis, Candida krusei, Aspergillus terreus, and Aconitum corymbiferum in Example 1, wherein ① represents Candida tropicalis, ② represents Candida krusei, ③ represents Aspergillus terreus, and ④ represents Aconitum corymbiferum.

[0037] Figure 1-11 、 1-12 The results are shown in Table 1 for the detection of Rhizopus stolonifer in Example 1.

[0038] Figure 1-13 、 1-14 These are the test results for Rhizomucor microti.

[0039] Figure 2-1 This is the detection result of Aspergillus fumigatus in Example 1.

[0040] Figure 2-2 This is the detection result of Candida parapsilosis in Example 1.

[0041] Figure 2-3 This is the detection result of Cryptococcus neoformans in Example 1.

[0042] Figure 2-4 These are the mixed tube test results for Aspergillus fumigatus, Candida parapsilosis, Cryptococcus neoformans and the internal reference in Example 1.

[0043] Figure 2-5 This is the detection result of Candida albicans in Example 1.

[0044] Figure 2-6 This is the detection result of the genus Transverse Mycoplasma (Transverse Mycoplasma umbellata) in Example 1.

[0045] Figure 2-7 These are the mixed tube test results of Candida albicans, Candida spp. (Candida corymbifera) and the internal reference in Example 1.

[0046] Figure 2-8 This is the detection result of Rhizomucor pusillus in Example 1.

[0047] Figure 2-9 The results of the detection of aflatoxin in Example 1 are shown in FIG.

[0048] Figure 2-10 This is the detection result of Candida tropicalis in Example 1.

[0049] Figure 2-11 These are the mixed tube test results of Rhizomucor pusillus, Aspergillus flavus, Candida tropicalis and the internal reference in Example 1.

[0050] Figure 2-12 This is the detection result of Candida glabrata in Example 1.

[0051] Figure 2-13 The results of the Aspergillus niger test in Example 1 are shown in FIG.

[0052] Figure 2-14 These are the mixed tube test results for Candida glabrata, Aspergillus niger, and the internal reference in Example 1.

[0053] Figure 2-15 The results are shown in Table 1 for the detection of Aspergillus terreus in Example 1.

[0054] Figure 2-16 This is the detection result of Candida krusei (Pichia kudrida) in Example 1.

[0055] Figure 2-17 This is the test result of Rhizopus stolonifer (black Rhizopus) in Example 1.

[0056] Figure 2-18 These are the mixed tube test results of Aspergillus terreus, Candida krusei (Pichia kudrivar), Rhizopus stolonifer (Black Rhizopus) and the internal reference in Example 1.

[0057] Figure 3-1 、 3-2 This is the detection result of Candida parapsilosis in Example 1.

[0058] Figure 3-3 、 3-4 This is the detection result of Cryptococcus neoformans in Example 1.

[0059] Figure 3-5 、 3-6 This is the detection result of Aspergillus fumigatus in Example 1.

[0060] Figure 3-7 、 3-8 It is the detection result of Candida albicans in Example 1.

[0061] Figure 3-9 、 3-10 This is the detection result of Pseudomonas aeruginosa in Example 1.

[0062] Figure 3-11 、 3-12 This is the detection result of Rhizomucor pusillus in Example 1.

[0063] Figure 3-13 、 3-14It is the detection result of Candida tropicalis in Example 1.

[0064] Figure 3-15 、 3-16 The results of the detection of aflatoxin in Example 1 are shown in FIG.

[0065] Figure 3-17 、 3-18 The test results of Aspergillus niger in Example 1 are shown in FIG.

[0066] Figure 3-19 、 3-20 The test results of Candida glabrata in Example 1 are shown in FIG.

[0067] Figure 3-21 、 3-22 This is the detection result of Candida krusei in Example 1.

[0068] Figure 3-23 、 3-24 This is the detection result of Aspergillus terreus in Example 1.

[0069] Figure 3-25 、 3-26 The results are shown in Table 1 for the detection of Rhizopus stolonifer in Example 1.

[0070] Figure 3-27 、 3-28 This is the detection result of the internal reference in Example 1.

[0071] Figure 4 The results are the repeatability test results on the CFX96 instrument in Example 1.

[0072] Figure 5 The results are the repeatability test results of Example 1 on the macrostone instrument.

[0073] Figure 6 This is the repeatability test result of experimenter 1 in Example 1.

[0074] Figure 7 This is the repeatability test result of experimenter 2 in Example 1.

[0075] Figure 8 This is the short-term stability test result of Example 1.

[0076] Figure 9 The long-term stability test results of Example 1 are shown. DETAILED DESCRIPTION

[0077] Example 1

[0078] 1. Primer and probe design

[0079] Targeting the fungi on the priority list of common clinical and pathogenic fungi, 13 fungus-specific primers and probes were designed and synthesized.

[0080] Table 1

[0081] Cryptococcus neoformans Candida glabrata Candida krusei Candida parapsilosis Candida tropicalis Candida albicans Aspergillus fumigatus Aspergillus flavus Aspergillus niger Aspergillus terreus Phytophthora umbellata Rhizopus stolonifer Rhizomucor microti

[0082] The multi-copy genes ITS1 and ITS2 in the fungal genome were selected. The ITS1 and ITS2 rDNA were obtained from the US NCBI database GenBank as the target rDNA gene sequence for detection. A common sequence was selected that could cover a certain gene sequence of fungi (such as Candida albicans). For example, all sequences of ITS1 and ITS2 rDNA of Candida albicans were compared using BLAST, and the variant sites were marked. Then, the ITS1 and ITS2 rRNA of Candida albicans were used as standards and input into the software PrimerPremier6 for primer and probe design. The designed probes and primers met the following conditions: (1) The GC content was between 40% and 50%; (2) The first base at the 5' end of the probe was not G; (3) The primer and probe did not have more than three consecutive G bases; (4) The probe sequence contained more C than G. A series of primers and probes were automatically generated, and the set of primers and probes with the lowest penalty and the least coverage of variant sites was selected as the optimal primers and probes for this study. For the variant sites on the primer probes, degenerated codes can be used to cover them. The designed primers and probes were compared on the NCBI database BLAST (http: / / blast.ncbi.nlm.nih.gov / Blast.Cgi) to determine their specificity. Primer-BLAST was used to perform computer simulation of the selected DNA region to evaluate the primer specificity. The evaluation included measuring the homodimer, heterodimer and hairpin formation of the primers for simultaneous detection, ensuring that the heterodimer delta G value of all primers was ≤-9kcal / mol, and confirming that there was no cross-reactivity with humans and non-target fungi.

[0083] The fungus-specific qPCR primer and probe sets used in the present invention were synthesized by Sangon Biotechnology.

[0084] Table 2

[0085]

[0086]

[0087]

[0088]

[0089] 2. Cultivate standard fungal strains for performance verification of the kit.

[0090] Table 3

[0091] Fungus Manufacturer No. Other numbers Cryptococcus neoformans VIP(WL)13019 ATCC32045 Candida glabrata VIP(L)13011 ATCC15126 Candida krusei ATCC14243 / Candida parapsilosis VIP(WL)10349 ATCC22019 Candida tropicalis VIP(B)11101 ATCC13803 Candida albicans VIP(WL)129001 ATCC90029 Aspergillus fumigatus VIP(N)10016 AS3.1320 Aspergillus flavus VIP(SN)16156 AS3.3950 Aspergillus niger VIP(LN)16588 AS3.3928 Aspergillus terreus VIP(SN)15941 AS3.3935 Rhizopus stolonifer VIP(JN)16301 AS3.31 Rhizomucor microti VIP(i)41653 XM-10(H).7 Phytophthora umbellata VIP(i)3161 /

[0092] 1) The strains used in this example were obtained from Xinyang Laiyao Biotechnology Co., Ltd. Fungal culture: Fungi were cultured on YM or YPD medium at 24° C. or 26° C. and incubated for 5-7 days.

[0093] 2) DNA extraction

[0094] Genomic DNA from all fungal cultures was extracted using a fungal genomic DNA extraction kit, and clinical samples were processed using Kangwei nucleic acid extraction and purification reagents. The total elution volume of DNA was 70 μL. DNA concentration was determined using Qubit (Thermo Fisher Scientific), and samples were stored at −20°C until analysis.

[0095] 3) Performance Verification

[0096] ① Real-time qPCR detection to determine the detection limit.

[0097] ② Primer specificity: Multiplex PCR primers were used for tNGS detection and SYBR qPCR method was used to verify primer specificity (amplification curve showed a typical S-shape and melting curve showed a single peak).

[0098] The dye method system and reaction procedure are as follows:

[0099] Table 4

[0100] Reagents 20μl system Mix 10 Forward Primer (10 μM) 0.2 Reverse Primer (10 μM) 0.2 DNA (10 ng) XμL <![CDATA[H2O]]> Addto20

[0101] Table 5

[0102]

[0103] Figure 1-1 、 1-2 The test results for Candida albicans are Figure 1-3 、 1-4 (One tube tests one fungus) These are the test results for Aspergillus flavus and Candida glabrata, where ① represents Aspergillus flavus and ② represents Candida glabrata. Figure 1-5 、 1-6 This is the test result of Aspergillus niger. Figure 1-7 、 1-8 (One tube tests one bacteria) These are the test results for Cryptococcus neoformans, Candida parapsilosis, and Aspergillus fumigatus, where ① represents Cryptococcus neoformans, ② represents Candida parapsilosis, and ③ represents Aspergillus fumigatus. Figure 1-9 、 1-10 (One tube tests one fungus) These are the test results for Candida tropicalis, Candida krusei, Aspergillus terreus, and Aconitum umbellatum. ① represents Candida tropicalis, ② represents Candida krusei, ③ represents Aspergillus terreus, and ④ represents Aconitum umbellatum. Figure 1-11 、 1-12 The test results for Rhizopus stolonifer are shown in Figure 2. Figure 1-13 、 1-14 These are the test results for Rhizomucor microti.

[0104] ③ Probe verification: qPCR verification was performed. If no S-shaped amplification curve appeared in the internal standard channel and an S-shaped amplification curve (Ct≤35) appeared in the FAM, HEX, or CY5 channel detection, the probe was determined to be able to specifically identify the corresponding fungal target.

[0105] To an eight-tube strip, add 12.5µl of mix, 0.5µl of each primer, 0.25µl of probe, and 10ng of DNA. Finally, add water to 25µl (reaction volume per tube). Mix thoroughly by pipetting. Perform 40-45 cycles of pre-denaturation at 95°C for 1 minute, denaturation at 95°C for 10 seconds, and annealing at 60°C for 30 seconds. Set up a single well for each bacterial strain. Then, mix 3-4 bacterial strains from each group in a single well. Set up a negative control tube.

[0106] Mixing pipe 1:

[0107] Table 6

[0108]

[0109] Figure 2-1 This is the test result of Aspergillus fumigatus. Figure 2-2 This is the detection result of Candida parapsilosis. Figure 2-3 The results of the Cryptococcus neoformans test are as follows: Figure 2-4 The results of mixed tube testing of the above three bacteria + internal reference are shown.

[0110] Mixer 2:

[0111] Table 7

[0112] strains Primer name aisle Dye method Single tube Mixed pipe Candida albicans CanAt-1 CY5 9.62 14.68 14.42 Helicobacterium genus Lic-1 FAM 21.53 23.06 22.01 Internal Reference GAPDHZ-1 TexasRed 23.26

[0113] Figure 2-5 This is the test result of Candida albicans. Figure 2-6 This is the test result of the genus Transverse Mycoplasma (Transverse Mycoplasma umbellata). Figure 2-7 This is the test result of the mixed tube of the above two bacteria + internal reference.

[0114] Mixer 3:

[0115] Table 8

[0116]

[0117]

[0118] Figure 2-8 This is the test result of Rhizomucor microti. Figure 2-9 The test results for aflatoxin are: Figure 2-10 This is the test result of Candida tropicalis. Figure 2-11 The results of mixed tube testing of the above three bacteria + internal reference are shown.

[0119] Mixer 4:

[0120] Table 9

[0121]

[0122] Figure 2-12 This is the test result of Candida glabrata. Figure 2-13 This is the test result of Aspergillus niger. Figure 2-14 This is the test result of the mixed tube of the above two bacteria + internal reference.

[0123] Mixer 5:

[0124] Table 10

[0125]

[0126] Figure 2-15 The test results for Aspergillus terreus are shown in Figure 2. Figure 2-16 This is the test result of Candida krusei (Pichia kudrida). Figure 2-17 This is the test result of Rhizopus stolonifer (black Rhizopus), Figure 2-18 The results of mixed tube testing of the above three bacteria + internal reference are shown.

[0127] ④ Probe specificity: Probe verification experiments were conducted on several fungi of the same genus but different species.

[0128] ⑤Sensitivity: minimum detection limit.

[0129] Quantitative sensitivity: Quantitative testing was performed using gradient dilution of the fungal reference product. The method was the same as that in the probe validation section. The standard curve had a good linear relationship with R^2>0.99. The results are shown in the table. Figures 3-1 to 3-28 .

[0130] Specific experimental procedure: 10 ng of each fungus group was mixed in a tube, and the mixture was filled with water to 35 μl. Then, 3.5 μl of the original mixed tube (10 ng) was transferred to another empty tube and the mixture was filled with water to 35 μl (10-fold dilution). This was repeated in a gradient dilution to 10^(-3) ng. Each gradient was performed in triplicate.

[0131] Table 11

[0132]

[0133] Repeatability: Repeatedly test the same sample concentration multiple times under the same experimental conditions, using the same method as in the probe validation section, and calculate the standard deviation and coefficient of variation (CV) of the results. Repeatedly test the fungus reference product at multiple concentration gradients, and calculate the standard deviation and coefficient of variation (CV) of the results. The coefficient of variation was within 1%.

[0134] Table 12

[0135]

[0136]

[0137] ⑦ Reproducibility: Repeat the test in different laboratories, with different operators and different instruments to verify the consistency of the results.

[0138] Two researchers with different levels of testing experience performed at least three replicate tests on the same batch of samples using two different instrument models. The reaction system and procedures were performed according to the probe method. The mean, standard deviation, and coefficient of variation (CV) of the test results for each researcher and instrument were calculated to assess the consistency of the results.

[0139] Human reproducibility 1: Experiments were performed on a CFX96 instrument. The results are shown in Tables 13 and Figure 4 .

[0140] Table 13

[0141]

[0142] Personnel repeatability 2: Experiments were performed on the Hongshi instrument. The results are shown in Table 14 and Figure 5 .

[0143] Table 14

[0144]

[0145] Instrument repeatability 1: The experiment was conducted by experimenter 1, and the results are shown in Table 15 and Figure 6 .

[0146] Table 15

[0147]

[0148] Instrument repeatability 2: The experiment was conducted by experimenter 2. The results are shown in Table 16 and Figure 7 .

[0149] Table 16

[0150]

[0151] ⑧Short-term stability: Performance test after short-term storage under different storage conditions (such as room temperature, 4°C, -20°C).

[0152] The reagents were stored at room temperature, 4°C, and -20°C for 1 day, 3 days, and 5 days before testing. The reaction system and procedure were carried out in accordance with the requirements of the above-mentioned probe method. The mean, standard deviation, and coefficient of variation (CV) of the test results under different storage conditions and storage times were calculated to evaluate the performance changes of the samples after short-term storage. The results are shown in Tables 17 and Figure 8 .

[0153] Table 17

[0154]

[0155]

[0156] ⑨Long-term stability: Performance test after long-term storage under recommended storage conditions.

[0157] The reagents were stored at -20°C for 2 years. The reaction system and procedure of the test were carried out according to the requirements of the above-mentioned probe method. The mean, standard deviation and coefficient of variation (CV) of the test results at different storage time points were calculated to evaluate the performance changes of the samples after long-term storage. The results are shown in Tables 18 and Figure 9 .

[0158] Table 18

[0159] 2023 2025 SE CV (%) Aspergillus fumigatus 25.45 24.50 0.67 2.69 Nearly smooth 22.46 25.65 2.26 9.38 New Hidden 24.08 26.19 1.49 5.94 Bai Nian 14.68 17.02 1.65 10.44 Umbrella branch 23.06 25.39 1.65 6.80 Rhizomucor 23.62 25.02 0.99 4.07 Aspergillus flavus 22.43 24.51 1.47 6.27 fervent thoughts 20.90 23.62 1.92 8.64 smooth 21.63 24.09 1.74 7.61 Aspergillus niger 31.30 31.92 0.44 1.39 Aspergillus terreus 23.01 25.41 1.70 7.01 Kerou 21.34 23.96 1.85 8.18 creeping roots 24.87 26.73 1.32 5.10

[0160] Example 2

[0161] This example provides a multiplex PCR kit for detecting respiratory pathogens, comprising reaction buffer, enzymes, primers, probes (internal reference & fungi, see Table 1 in Example 1), positive and negative controls (ddH2O), and other components. The design principle is based on fluorescent PCR-probe technology, utilizing specific primers and probes to identify and quantify specific gene sequences of target fungi.

[0162] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A primer and probe for multiplex PCR detection of respiratory pathogens, characterized in that: include: Primers of the nucleotide sequences shown in SEQ ID NOs. 5-6 and a probe of the nucleotide sequence shown in SEQ ID NO. 4 for detecting Candida parapsilosis; Primers of the nucleotide sequences shown in SEQ ID NOs. 8-9 and a probe of the nucleotide sequence shown in SEQ ID NO. 7 for detecting Cryptococcus neoformans; Primers of the nucleotide sequences shown in SEQ ID NOs. 11-12 and a probe of the nucleotide sequence shown in SEQ ID NO. 10 for detecting Aspergillus fumigatus; Primers of the nucleotide sequences shown in SEQ ID NOs. 14-15 and a probe of the nucleotide sequence shown in SEQ ID NO. 13 for detecting Candida albicans; Primers of the nucleotide sequences shown in SEQ ID NOs. 17-18 and a probe of the nucleotide sequence shown in SEQ ID NO. 16 for detecting Pseudomonas aeruginosa; Primers of the nucleotide sequences shown in SEQ ID NOs. 20-21 and a probe of the nucleotide sequence shown in SEQ ID NO. 19 for detecting Rhizomucor pusillus; Primers of the nucleotide sequences shown in SEQ ID NOs. 23-24 and a probe of the nucleotide sequence shown in SEQ ID NO. 22 for detecting Candida tropicalis; Primers of the nucleotide sequences shown in SEQ ID NOs. 26-27 and a probe of the nucleotide sequence shown in SEQ ID NO. 25 for detecting Aspergillus flavus; Primers of the nucleotide sequences shown in SEQ ID NOs. 29-30 and a probe of the nucleotide sequence shown in SEQ ID NO. 28 for detecting Aspergillus niger; Primers of the nucleotide sequences shown in SEQ ID NOs. 32-33 and a probe of the nucleotide sequence shown in SEQ ID NO. 31 for detecting Candida glabrata; Primers of the nucleotide sequences shown in SEQ ID NOs. 35-36 and a probe of the nucleotide sequence shown in SEQ ID NO. 34 for detecting Candida krusei; Primers of the nucleotide sequences shown in SEQ ID NOs. 38-39 and a probe of the nucleotide sequence shown in SEQ ID NO. 37 for detecting Aspergillus terreus; Primers of the nucleotide sequences shown in SEQ ID NO. 41-42 and a probe of the nucleotide sequence shown in SEQ ID NO. 40 for detecting Rhizopus stolonifer.

2. Use of the primers and probes according to claim 1 in preparing products for detecting respiratory pathogens.

3. The use according to claim 2, characterized in that The product is a reagent or a kit.

4. A product for detecting respiratory pathogens, characterized in that: The product contains the primers and probes described in claim 1.

5. The product according to claim 4, characterized in that The product is a reagent or a kit.

6. The product according to claim 5, characterized in that The kit also includes a detection reagent and a nucleic acid extraction reagent.

7. The product according to claim 6, characterized in that The detection reagents include sterile water and PCR Mix.

8. The product according to any one of claims 4 to 7, characterized in that The product also contains primers and probes for detecting internal reference genes.

9. The product according to claim 8, characterized in that The primer sequences for detecting the internal reference gene are shown in SEQ ID NO. 2-3, and the probe sequence is shown in SEQ ID NO. 1.

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