A primer set, probe and kit for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus

Abstract

The present invention provides a primer set, a probe and a kit for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus. The primer set includes: a primer set for detecting Aspergillus fumigatus abaA gene, whose nucleotide sequence is shown in SEQ ID NOs: 1-2; a primer set for detecting Aspergillus niger ph1 gene, whose nucleotide sequence is shown in SEQ ID NOs: 25-26; a primer set for detecting Aspergillus flavus taa gene, whose nucleotide sequence is shown in SEQ ID NOs: 43-44. The probe includes a probe for detecting Aspergillus fumigatus abaA gene, whose nucleotide sequence is shown in SEQ ID NO: 3; a probe for detecting Aspergillus niger ph1 gene, whose nucleotide sequence is shown in SEQ ID NO: 27; and a probe for detecting Aspergillus flavus taa gene, whose nucleotide sequence is shown in SEQ ID NO: 45. The primers and probes can specifically distinguish Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus.

Description

Technical Field

[0001] The invention relates to the technical field of gene detection, and in particular to a primer set, a probe and a kit thereof for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus. Background Art

[0002] Invasive fungal disease (IFD) refers to fungal infections caused by fungi invading human subcutaneous mucosal tissues, blood, and internal organs. In recent years, invasive fungal diseases have gradually become serious infectious diseases and are one of the most serious threats to human health and life. With the widespread use of invasive examinations, broad-spectrum antibiotics, immunosuppressants, glucocorticoids, and tumor chemotherapy drugs, the risk of fungal infection has also increased. Therefore, early detection and identification of pathogen types are particularly important for clinical diagnosis and treatment. In addition, the pathogenic spectrum of major pathogenic fungi has been changing in recent years. In addition to common fungal infections that have been recognized by humans for many years, many environmental fungi that were not pathogenic in the past have evolved and mutated, gradually becoming pathogenic bacteria, and even becoming multidrug-resistant strong pathogens (for example, Candida auris discovered in recent years is considered a super fungus). Because the clinical manifestations and symptoms of invasive fungal diseases are atypical and are often masked by underlying diseases, their early diagnosis mainly relies on traditional diagnostic methods such as direct microscopy, culture, and imaging.

[0003] Histopathological examination is the gold standard for the diagnosis of invasive fungal infection. Through histopathological examination, the species and genus of fungi, such as Cryptococcus, Candida, Aspergillus, and pigmented fungi, can be determined. Fungal examination can basically be divided into two categories: culture techniques and non-culture techniques. Non-culture techniques are further divided into microscopy techniques, biochemical immunoassay techniques, and molecular biology techniques. The former is to directly observe the morphology of fungi; the latter is to detect the antigens, antibodies, and nucleic acids of fungi. The combined use of these techniques can greatly improve the accuracy of the diagnosis of invasive fungal infection. Among them, direct smear staining and microscopic examination of specimens and inoculation on culture media for isolation and culture are common methods for diagnosing pathogenic microorganisms of fungal infectious diseases. However, it has the following disadvantages: 1) The culture time is long, and delayed diagnosis leads to increased mortality. For example, Candida can be cultured and identified within 48 hours, while the culture time of Cryptococcus can be up to 2-4 weeks. If the treatment of invasive Candida is delayed for 4 days, the mortality rate will increase from 15.4% to 41.4%; 2) It is difficult to distinguish between colonization and infection. Since common fungi such as Candida and Aspergillus are both conditionally pathogenic bacteria that colonize the normal human respiratory tract, urinary tract, etc., if the specimen is taken from a non-sterile site, culture and microscopy cannot distinguish between colonization and infection; 3) Patient compliance is poor due to the invasive nature of the examination. If an invasive examination is performed to obtain the sample to be tested from a sterile site, it is difficult to implement, especially for patients with underlying diseases such as pulmonary aspergillosis; 4) It is difficult to identify the fungus species based on morphology. It lacks sensitivity and accuracy. For example, hyphae of the same fungus species will show different morphologies under the microscope. Traditional pathogenic microorganism diagnostic methods are cumbersome to operate, have a long detection cycle, low specificity of results, and require a relatively high level of technical skills from the operator, which limits the clinical application of traditional pathogenic microorganism diagnostic methods in the diagnosis of invasive fungal diseases.

[0004] In recent years, serological diagnostic methods have been gradually applied to the early diagnosis of invasive fungal diseases, becoming an important supplement and improvement to traditional diagnostic methods. Serological diagnostic techniques for invasive fungal diseases can be roughly divided into two categories: antigen detection and antibody detection. Antigen detection mainly includes G test, GM test, GXM test and Mn test, etc. Antibody detection mainly refers to the detection of IgM and IgG antibodies specific to each genus. For patients with normal immune function, the results of antibody detection can better reflect the overall stage of disease development and have greater detection value. Serological diagnosis is easy to carry out and easy to operate, and the results between laboratories are highly comparable, which makes up for the shortcomings of traditional diagnostic technology.

[0005] With the continuous advancement of molecular biology technology, molecular diagnosis has brought new opportunities for the diagnosis of clinical fungal diseases. Compared with traditional methods and serological detection methods, molecular diagnostic methods represented by PCR and constant temperature amplification have been widely promoted and applied due to their high sensitivity, high sensitivity, specificity and lack of restrictions.

[0006] Real-time fluorescence quantitative PCR (Quantitative Real-time PCR) nucleic acid detection technology does not require culture and has been shown to help expand the range of detectable pathogens and improve the detection rate. It is suitable for detecting low-abundance fungal DNA in clinical specimens and has good application prospects for the detection of Aspergillus, Pneumocystis and Candida. The European Organization for Research and Treatment of Cancer (EORTC) and the Fungal Diseases Study Group Education and Research Consortium (MSGERC) have included it in the consensus definition of invasive fungal disease (2020) as one of the diagnostic methods for invasive fungal disease. In 2022, the fungal clinical medicine community took the lead in drafting the "Expert Consensus on the Clinical Application of Laboratory Diagnostic Methods for Invasive Fungal Diseases", which for the first time wrote fluorescent quantitative PCR for fungal nucleic acid detection technology and used it as a recommended detection method; in March of that year. The National Medical Products Administration convened manufacturers engaged in fungal research to participate in the formulation of the "Guidelines for Registration and Review of Aspergillus Nucleic Acid Detection Reagents". QPCR samples are easy to obtain, non-invasive and repeatable. Sputum is the most common, non-invasive, easily collected and repeatedly sample source for respiratory diseases and can be used to detect pulmonary fungal infections. There are related reports that Aspergillus isolated from sputum and Aspergillus isolated from bronchoalveolar lavage fluid are homologous, which suggests that the microbiological evidence obtained from sputum and bronchoalveolar lavage fluid has similar diagnostic capabilities for pulmonary aspergillosis. Although sputum batches are susceptible to differences caused by patient factors, such as whether each sampling can be carried out according to the standard process, and it is a complex sample itself, and various impurities can affect the test results, sputum samples are easy to obtain non-invasively, if the detection technology is sensitive, stable and reliable enough, sputum samples can be used as a sample source for molecular biological methods to detect fungal infections and assist clinical diagnosis.

[0007] Currently, molecular diagnosis represented by real-time fluorescence quantitative PCR and related technologies has gradually become an important method for diagnosing infectious diseases. It is not only of great significance for early initiation of antifungal treatment, improving patient prognosis and reducing mortality, but also has great potential for auxiliary diagnosis of invasive fungal diseases. Summary of the invention

[0008] The embodiment of the present invention provides a primer set, a probe and a kit thereof for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus, so as to solve at least one of the problems existing in the related art. To achieve this object, the present invention is implemented by the following technical solutions.

[0009] A primer set for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus, comprising: a primer set for detecting the abaA gene of Aspergillus fumigatus, whose nucleotide sequence is shown in SEQ ID NOs: 1-2; a primer set for detecting the ph1 gene of Aspergillus niger, whose nucleotide sequence is shown in SEQ ID NOs: 25-26; and a primer set for detecting the taa gene of Aspergillus flavus, whose nucleotide sequence is shown in SEQ ID NOs: 43-44.

[0010] A probe for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus, comprising: a probe for detecting the abaA gene of Aspergillus fumigatus, whose nucleotide sequence is shown in SEQ ID NO:3; a probe for detecting the ph1 gene of Aspergillus niger, whose nucleotide sequence is shown in SEQ ID NO:27; and a probe for detecting the taa gene of Aspergillus flavus, whose nucleotide sequence is shown in SEQ ID NO:45.

[0011] The present invention is directed to the specific genes abaA gene, GliT gene, cyp51A gene, and msh6 gene of Aspergillus fumigatus; the specific genes aglU gene, nad5 gene, bgl1 gene, ph1 gene, and glkA gene of Aspergillus niger; and the specific genes xyn gene, aflD gene, alk1 gene, aflM gene, and taa gene of Aspergillus flavus, and primer probes are designed respectively, and real-time fluorescence quantitative PCR is used for detection. The results show that the primer probe using the abaA gene of Aspergillus fumigatus as a target, the primer probe using the ph1 gene of Aspergillus niger as a target, and the primer probe using the taa gene of Aspergillus flavus as a target have better detection effects. The target quality is higher, and the specificity and sensitivity are better.

[0012] Preferably, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0013] Preferably, the fluorescent reporter groups include FAM, ROX, and CY5; and the fluorescent quencher groups include BHQ1, BHQ2, and BHQ3.

[0014] The primer set and probe are used in preparing a kit for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus.

[0015] A kit for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus contains the above primer set and probe.

[0016] Preferably, the kit further contains a multiplex PCR reaction solution.

[0017] Preferably, the reaction solution of the multiplex PCR includes a PCR buffer, a DNA polymerase, and dNTPs (dATP, dTTP, dCTP, dGTP, dUTP).

[0018] Preferably, the kit further contains primers and probes for detecting an internal reference gene (human RNase P gene), the nucleotide sequences of the primers are shown in SEQ ID NOs:46-47, and the nucleotide sequence of the probe is shown in SEQ ID NO:48.

[0019] Preferably, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group; the fluorescent reporter group is HEX; and the fluorescent quencher group is BHQ1.

[0020] A method for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus for non-disease diagnosis purposes, using the DNA of a sample to be tested as a template, performing PCR using the above primer set and probe, wherein the probes are respectively labeled with different fluorescences, and when an obvious S-shaped amplification curve appears in the fluorescence channel corresponding to only the probe for detecting the Aspergillus fumigatus abaA gene and the Ct value is ≤36, the sample to be tested is judged to be positive for Aspergillus fumigatus, and negative for Aspergillus niger and Aspergillus flavus;

[0021] When only the fluorescence channel corresponding to the probe detecting the ph1 gene of Aspergillus niger shows an obvious S-shaped amplification curve and the Ct value is ≤36, the sample to be tested is judged to be positive for Aspergillus niger, and negative for Aspergillus fumigatus and Aspergillus flavus;

[0022] When an obvious S-type amplification curve appears only in the fluorescence channel corresponding to the probe detecting the Aspergillus flavus taa gene and the Ct value is ≤35, the sample to be tested is judged to be positive for Aspergillus flavus and negative for Aspergillus fumigatus and Aspergillus niger.

[0023] Preferably, the PCR reaction system is: 4 μL buffer, 1 μL DNA enzyme, 0.4 μL dNTP, 7.6-11.6 μL water, 2 μL of the mixture of the primer set and the probe, 5 μL template, and the total volume is 20 μL; the reaction procedure is: 95°C for 3 min, 1 cycle; 95°C for 5-15 s, 56-60°C for 30 s, and 40-45 cycles; the volume ratio of the primer-probe mixture of Aspergillus fumigatus, the primer-probe mixture of Aspergillus niger, and the primer-probe mixture of Aspergillus flavus in the mixture is 1:(0.25-1):(0.25-1), the volume ratio of primers to probes in the primer-probe mixture is 2:(0.25-1), the final concentration of the primers is 10 μM; the final concentration of the probe is 2.5-10 μM.

[0024] Preferably, the PCR reaction system is: 4 μL buffer, 1 μL DNA enzyme, 0.4 μL dNTP, 7.6 μL water, 2 μL of the mixture of the primer set and the probe, 5 μL template, and a total volume of 20 μL; the reaction program is: 95°C for 3 min, 1 cycle; 95°C for 10 s, 58°C for 30 s, and 40 cycles.

[0025] Preferably, the volume ratio of the primer-probe mixture of Aspergillus fumigatus, the primer-probe mixture of Aspergillus niger and the primer-probe mixture of Aspergillus flavus in the mixture is 1:0.5:0.5, and the volume ratio of primers to probes in the primer-probe mixture is 2:1.

[0026] The embodiments of the present invention have the following beneficial effects:

[0027] The present invention designs primer and probe sequences for Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus in sputum samples, and performs fluorescence quantitative PCR detection. By using the primers and probes of the present invention, the three Aspergillus species, Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus, can be specifically distinguished, thereby performing targeted medication, accelerating the treatment process, improving the treatment effect, ensuring patient safety, reducing adverse reactions, controlling medical costs, and promoting the healthy development of the pharmaceutical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0029] Figure 1 This is the amplification curve corresponding to different primer probes of Aspergillus fumigatus (FAM channel: blue).

[0030] Figure 2 This is the amplification curve corresponding to different primer probes of Aspergillus niger (ROX channel: orange).

[0031] Figure 3 This is the amplification curve corresponding to different primer probes of Aspergillus flavus (CY5 channel: red).

[0032] Figure 4 This is the amplification curve corresponding to the primer-probe combination with a volume ratio of 1:1:1:1 for fum:IC:fla:niger.

[0033] Figure 5 These are the amplification curves corresponding to the primer-probe combinations with volume ratios of fum:IC:fla:niger of 1:1:0.5:0.5 and 1:1:0.25:0.25.

[0034] Figure 6 Shows the amplification curves corresponding to different template amounts.

[0035] Figure 7 Shows the amplification curves corresponding to different dNTP addition amounts.

[0036] Figure 8 These are the test results of Aspergillus fumigatus primer probe fum-abaA detecting nucleic acid of Aspergillus niger and Aspergillus flavus quality control products.

[0037] Fig. 9 These are the test results of Aspergillus niger primer probe niger-ph1 detecting nucleic acid of Aspergillus fumigatus and Aspergillus flavus quality control products.

[0038] Fig.10 These are the test results of the Aspergillus fumigatus and Aspergillus niger quality control nucleic acid detected by the Aspergillus fumigatus primer probe fla-taa. DETAILED DESCRIPTION

[0039] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. However, it will be appreciated by those skilled in the art that in the embodiments of the present invention, many technical details are proposed in order to enable the reader to better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical scheme claimed in the present application can be implemented. The division of the following embodiments is for the convenience of description, and should not constitute any limitation on the specific implementation of the present invention. The various embodiments can be combined and referenced with each other without contradiction.

[0040] Example 1 Design of primer probe

[0041] 1. First, the specific genes of Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus were searched through the literature and the gene sequences were downloaded. The specific genes of Aspergillus fumigatus include abaA gene (AY772088.1), GliT gene (XM_745770.1), cyp51A gene (MW788327.1), and msh6 gene (XM_746902.1); the specific genes of Aspergillus niger include aglU gene (XM_001402016.3), nad5 gene (DQ178137.1 ), bgl1 gene (XM_001398779.3), ph1 gene (PP621893.1), glkA (XM_001395875.3) gene; the specific genes of Aspergillus flavus include: xyn gene (AY495911.1), aflD gene (PP493380.1), alk1 gene (AF324246.1), aflM gene (FN398175.1), taa gene (AY495865.1). Then, specific primer probe sequences were designed for each of the above gene sequences manually and by software Oligo 7, and primer probes were designed for human RNase P gene (internal reference, IC). The sequences of all the above specific primers (F, R) and probes (P) are shown in Table 1 below.

[0042] Table 1

[0043]

[0044]

[0045] Example 2 Test of the effects of different primers and probes on three fungi

[0046] 1. Extraction of nucleic acid from samples to be tested

[0047] Clinical sputum was taken as the test sample, and the sample was tested by Aspergillus, Cryptococcus neoformans and Pneumocystis jirovecii nucleic acid detection kit (Zhuocheng Huisheng) to determine that it contained Aspergillus.

[0048] Extract the nucleic acid (DNA) of the sample to be tested by thermal lysis method, draw 500 μL of the sample to be tested into a 1.5mL centrifuge tube, thermally lyse at 95℃ for 10min; then centrifuge at 12000rpm for 1min; draw the supernatant into a new 1.5mL centrifuge tube, add 30 μL of magnetic beads, shake for 10min, centrifuge at 12000rpm for 1min, place on a magnetic rack, let stand for 2min, and discard the supernatant; add 500 μL of washing solution (75% ethanol), shake for 2min, centrifuge for 30s, place on a magnetic rack, let stand for 2min, and discard the supernatant; repeat the above steps once; finally add the extraction solution (TE solution), shake for 5min, centrifuge for 30s, place on a magnetic rack, let stand for 2min, draw the supernatant (be careful not to suck up the magnetic beads) into a new 1.5mL centrifuge tube to obtain the nucleic acid extract of the sample to be tested, and mark it.

[0049] 2. PCR testing

[0050] The extracted nucleic acid extract was used as a template, and the primer probes of Example 1 were used for PCR reaction. The volume ratios of the primer probes in the primer probe mixture (F:R:P) were 1:1:1, 1:1:0.75, 1:1:0.5, and 1:1:0.25, respectively. The initial concentrations of the components F, R, and P were all 100 μM. 10 μL of F, 10 μL of R, 10 μL of P (7.5, 5, 2.5), and 70 μL of sterile water (72.5, 75, 77.5) were used to form a single primer probe mixture. The final volume was 100 μL, and the final concentrations of the components were F:R:P=10 μM:10 μM:2.5-10 μM. The reaction system of the PCR reaction is shown in Table 2 below, and the reaction procedure is shown in Table 3 below.

[0051] Table 2

[0052]

[0053] Table 3

[0054]

[0055] 3. Results

[0056] (1) The test results corresponding to different primer probes of Aspergillus fumigatus are shown in Table 4 below. The amplification curves corresponding to different primer probes of Aspergillus fumigatus are shown in Table 4 below. Figure 1 As shown,

[0057] Table 4

[0058]

[0059]

[0060] Table 4 and Figure 1 The results showed that the primer probe with a clear S-shaped curve, with a smaller Ct value and a larger Rn value, was better, according to the Ct value and Rn value. According to the chart, the primer probe designed with the abaA gene of Aspergillus fumigatus as the target was better.

[0061] (2) The test results corresponding to different primer probes of Aspergillus niger are shown in Table 5 below. The amplification curves corresponding to different primer probes of Aspergillus niger are shown in Table 5 below. Figure 2 As shown,

[0062] Table 5

[0063]

[0064]

[0065] Table 5 and Figure 2 The results showed that the primer probe with a clear S-shaped curve, with a smaller Ct value and a larger Rn value, was better, according to the Ct value and Rn value. According to the chart, the primer probe designed with the ph1 gene of Aspergillus niger as the target was better.

[0066] (3) The test results corresponding to different primer probes of Aspergillus flavus are shown in Table 6 below. The amplification curves corresponding to different primer probes of Aspergillus flavus are shown in Table 6 below. Figure 3 As shown,

[0067] Table 6

[0068]

[0069] Table 6 and Figure 3 The results showed that the primer probe with a clear S-shaped curve, with a smaller Ct value and a larger Rn value, was better, based on the size of the Ct value and the Rn value. According to the chart, the primer probe designed with the taa gene of Aspergillus flavus as the target was better.

[0070] Example 3 Enzyme Screening

[0071] 1. Sample

[0072] Clinical sputum samples were taken as test samples and numbered No. 1 and No. 2 respectively. These two samples were tested with Aspergillus, Cryptococcus neoformans and Pneumocystis jirovecii nucleic acid detection kits (Zhuocheng Huisheng) to determine that they contained Aspergillus.

[0073] DNA polymerases from 12 different manufacturers or mixtures containing DNA polymerases (Mix): (1) Global FKQ3001, (2) Yisheng C55P1, (3) Yisheng 13891 (UT), (4) Novozymes, (5) Quanshijin, (6) Tiangen, (7) Global HQMU3001, (8) Global KQMU3001, (9) Yisheng Lyo-Ready, (10) Takara, (11) Yisheng C198P1, (12) Yisheng 16710 (PUT).

[0074] 2. PCR

[0075] According to the nucleic acid extraction method of Example 2, nucleic acids of the test samples No. 1 and No. 2 were extracted to obtain corresponding nucleic acid extracts. The extracted nucleic acid extracts were used as templates, and PCR reactions were performed using primers (fla-taa-F / fla-taa-R) and probes (fla-taa-P) designed based on the taa gene of Aspergillus flavus in Example 1. The volume ratio of primers to probes in the primer / probe mixture (Primer / Probe Mix) was 1:1:1.

[0076] The reaction systems and reaction procedures for PCR experiments using enzymes from 12 different manufacturers are as follows:

[0077] (1) The reaction system and reaction procedure corresponding to Global FKQ3001 are shown in Tables 7 and 8 below.

[0078] Table 7

[0079]

[0080] Table 8

[0081]

[0082] (2) The reaction system and reaction procedure corresponding to Yisheng C55P1 are shown in Tables 9 and 10 below.

[0083] Table 9

[0084]

[0085] Table 10

[0086]

[0087] (3) The reaction system and reaction procedure corresponding to Yisheng 13891 (UT) are shown in Tables 11 and 12 below.

[0088] Table 11

[0089]

[0090]

[0091]

[0092] (4) The reaction system and reaction procedure corresponding to Novozyme are shown in Tables 13 and 14 below.

[0093] Table 13

[0094]

[0095] Table 14

[0096]

[0097] (5) The reaction system and reaction procedure corresponding to the full formula are shown in Tables 15 and 16 below.

[0098] Table 15

[0099]

[0100] Table 16

[0101]

[0102] (6) The reaction system and reaction procedure corresponding to Tiangen are shown in Tables 17 and 18 below.

[0103] Table 17

[0104]

[0105] Table 18

[0106]

[0107] (7) The reaction system and reaction procedure corresponding to Global HQMU3001 are shown in Tables 19 and 20.

[0108] Table 19

[0109]

[0110] Table 20

[0111]

[0112] (8) The reaction system and reaction procedure corresponding to Global KQMU3001 are shown in Tables 21 and 22.

[0113] Table 21

[0114]

[0115] Table 22

[0116]

[0117] (9) The reaction system and reaction procedure corresponding to Yisheng Lyo-Ready are shown in Tables 23 and 24 below.

[0118] Table 23

[0119]

[0120] Table 24

[0121]

[0122] (10) Takara's corresponding reaction system and reaction procedure are shown in Tables 25 and 26.

[0123] Table 25

[0124]

[0125] Table 26

[0126]

[0127]

[0128] (11) The reaction system and reaction procedure corresponding to Yisheng C198P1 are shown in Tables 27 and 28 below.

[0129] Table 27

[0130]

[0131] Table 28

[0132]

[0133] (12) The reaction system and reaction procedure corresponding to Yisheng 16710 (PUT) are shown in Tables 29 and 30 below.

[0134] Table 29

[0135]

[0136] Table 30

[0137]

[0138] 3. Results

[0139] The PCR results corresponding to enzymes from 12 different manufacturers are shown in Tables 31 and 32 below, where the negative control is normal saline, divided into two samples, labeled No. 1 and No. 2.

[0140] Table 31

[0141]

[0142]

[0143] Table 32

[0144]

[0145]

[0146] According to the Ct values ​​and fluorescence (Rn) values ​​in Tables 31 and 32, the primer probe with a smaller Ct value and a larger Rn value and an obvious S-shaped curve is better. Among them, the enzymes of Global FKQ3001 and Yishen 16710 (PUT) performed better, and Global FKQ3001 was selected for subsequent experiments.

[0147] Example 4 Screening of multiple probes

[0148] The single probes screened in Example 2 were subjected to combination screening at different ratios, and the primer probes of each single target were mixed at a volume ratio of F:R:P=1:1:1 to obtain a primer probe mixture, and the volume ratios of Aspergillus fumigatus primer probe mixture (fum): human internal reference primer probe mixture (IC): Aspergillus flavus primer probe mixture (fla): Aspergillus niger primer probe mixture (niger) were 1:1:1:1, 1:1:0.5:0.5, and 1:1:0.25:0.25 to obtain a mixture of primers and probes. The nucleic acid extract of the sample determined to contain Aspergillus by the kit was used as a template, and qPCR was performed using a mixture of primers and probes at different ratios. The 5' end of the probe fum-abaA-P was labeled with FAM fluorescence, and the 3' end was labeled with BHQ1 fluorescence; the 5' end of the probe IC-RNase PP was labeled with HEX fluorescence, and the 3' end was labeled with BHQ1 fluorescence; the 5' end of the probe niger-ph1-P was labeled with ROX fluorescence, and the 3' end was labeled with BHQ2 fluorescence; the 5' end of the probe fla-taa-P was labeled with CY5 fluorescence, and the 3' end was labeled with BHQ3 fluorescence. The reaction system used was: 4μL buffer, 1μL enzyme, 1μL dNTP, 7μL water, 2μL primer probe mixture, 5μL template, and the total volume was 20μL. The reaction program was: 95℃3min, 1 cycle; 95℃30s, 58℃30s, 45 cycles.

[0149] The results corresponding to primer-probe combinations with different ratios are shown in Table 33 below. In the table, the full target includes the abaA gene of fum, the RNase P gene of IC, the taa gene of fla, and the ph1 gene of niger. The amplification curve corresponding to the primer-probe combination with a volume ratio of fum: IC: fla: niger of 1:1:1:1 is shown in Figure 4 As shown in FIG. 1 , the amplification curves corresponding to the primer-probe combinations with a volume ratio of fum:IC:fla:niger of 1:1:0.5:0.5 and 1:1:0.25:0.25 are shown in FIG. Figure 5 As shown,

[0150] Table 33

[0151]

[0152] According to Table 33 and Figure 4 and Figure 5 The results show that the primer probe with a smaller Ct value and a larger Rn value and an obvious S-shaped curve is better. As shown in the chart, a volume ratio of fum:IC:fla:niger of 1:1:0.5:0.5 is better.

[0153] Example 5 Screening of procedures and systems

[0154] The nucleic acid extract of the sample determined to contain Aspergillus by the kit was used as a template, and qPCR was performed using a multiple primer probe with a volume ratio of fum:IC:fla:niger of 1:1:0.5:0.5 (wherein the F:R:P of each single primer probe is 1:1:1). The 5' end of the probe fum-abaA-P was fluorescently labeled with FAM, and the 3' end was fluorescently labeled with BHQ1; the 5' end of the probe IC-RNase PP was fluorescently labeled with HEX, and the 3' end was fluorescently labeled with BHQ1; the 5' end of the probe niger-ph1-P was fluorescently labeled with ROX, and the 3' end was fluorescently labeled with BHQ2; the 5' end of the probe fla-taa-P was fluorescently labeled with CY5, and the 3' end was fluorescently labeled with BHQ3. The following experiment was performed according to the reaction procedure and reaction system of Example 4. Physiological saline was used as a control.

[0155] 1. Screening of template quantity

[0156] The template amounts were 5, 3, and 1 μL, respectively, and the reaction system was the same as in Example 4. When the template amount was 3 μL (or 1 μL), only the volume of water was adjusted to 9 μL (or 11 μL) to keep the total volume unchanged.

[0157] The detection results corresponding to different template amounts are shown in Table 34 below. The full targets in the table include the abaA gene of fum, the RNase P gene of IC, the taa gene of fla, and the ph1 gene of niger. The amplification curves corresponding to different template amounts are shown in Figure 6 As shown,

[0158] Table 34

[0159]

[0160] According to Table 34 and Figure 6 From the Ct value and fluorescence value (Rn), it can be seen that the effect is better when the template amount is 5 μL.

[0161] 2. Screening of dNTP addition amount

[0162] The amount of dNTP added was 0.4 μL and 1 μL respectively, and the reaction system was the same as in Example 4. When the amount of dNTP added was 0.4 μL, the volume of water was only adjusted to 7.6 μL, so that the total volume remained unchanged. The reaction procedure was the same as in Example 4.

[0163] The detection results corresponding to different dNTP addition amounts are shown in Table 35 below. The full targets in the table include the abaA gene of fum, the RNase P gene of IC, the taa gene of fla, and the ph1 gene of niger. The amplification curves corresponding to different dNTP addition amounts are shown in Table 35. Figure 7 As shown,

[0164] Table 35

[0165]

[0166] According to Table 35 and Figure 7 From the Ct value and fluorescence value (Rn), it can be seen that the data of dNTP addition amount of 0.4μL is better than that of dNTP addition amount of 1μL, so dNTP addition amount of 0.4μL is selected.

[0167] 3. Screening of reaction procedures

[0168] (1) Selection of cycle number

[0169] The cycle numbers were 45 and 40 respectively. The reaction procedure was the same as in Example 4, except that the cycle number 45 was changed to 40.

[0170] The test results corresponding to different cycle numbers are shown in Table 36 below.

[0171] Table 36

[0172]

[0173]

[0174] Judging from the Ct value and fluorescence value (Rn) in Table 36, and considering the Ct value of the negative control, the cycle number was selected as 40.

[0175] (2) Screening of annealing temperature. This indicator is related to the specificity issue.

[0176] The annealing temperatures were 58°C, 60°C, and 56°C, respectively. The reaction procedure was the same as in Example 4, except that the number of cycles was 40 and the annealing temperature of 58°C was changed to 60°C and 56°C.

[0177] The test results corresponding to the annealing temperatures of 60°C and 56°C are shown in Table 37 below.

[0178] Table 37

[0179]

[0180] Judging from the Ct values ​​and fluorescence values ​​in Tables 36 and 37, the annealing temperature is better at 58°C.

[0181] (3) Screening of denaturation time

[0182] The denaturation times were 5 s, 10 s, and 15 s, respectively. The reaction procedure was the same as in Example 4, except that the number of cycles was 40 and the denaturation time of 30 s was changed to 5 s, 10 s, and 15 s.

[0183] The test results corresponding to different denaturation times are shown in Table 38 below.

[0184] Table 38

[0185]

[0186]

[0187] According to the Ct values ​​and fluorescence (Rn) values ​​in Tables 36 and 38, the final denaturation time selected was 10 s.

[0188] In summary, when the primer probe of the present invention is used for PCR detection, the reaction system used is: 4 μL buffer, 1 μL enzyme, 0.4 μL dNTP, 7.6 μL water, 2 μL primer probe mixture, 5 μL template, and the total volume is 20 μL; the reaction procedure is: 95°C 3min, 1 cycle; 95°C 10s, 58°C 30s, 40 cycles, and the detection results have higher specificity and sensitivity.

[0189] Example 6 Cross-reaction

[0190] The single probes selected in Example 2, namely fum-abaA (F:R:P=1:1:1), niger-ph1 (F:R:P=1:1:1), and fla-taa (F:R:P=1:1:1), were used to extract nucleic acids from Aspergillus fumigatus (BNCC363594), Aspergillus niger (BNCC363591), and Aspergillus flavus (BNCC368285) liquid indoor quality control products purchased from Beina Biological as templates, and a combination of a certain primer probe and the remaining two target nucleic acids was used to detect whether there was a cross reaction between the three primer probes. The reaction system was: 4 μL buffer, 1 μL enzyme, 0.4 μL dNTP, 7.6 μL water, 2 μL primer probe mixture, 5 μL template, and a total volume of 20 μL; the reaction procedure was: 95°C 3min, 1 cycle; 95°C 10s, 58°C 30s, and 40 cycles.

[0191] The results of the Aspergillus fumigatus primer probe fum-abaA detection of Aspergillus niger and Aspergillus flavus quality control nucleic acid are as follows Figure 8 As shown in Figure 1, the results of the Aspergillus niger primer probe niger-ph1 detecting nucleic acids of Aspergillus fumigatus and Aspergillus flavus quality control products are as follows: Fig. 9 As shown in Figure 1, the results of the Aspergillus fumigatus and Aspergillus niger quality control nucleic acid detection using the Aspergillus fumigatus primer probe fla-taa are as follows: Fig.10 shown.

[0192] Figure 8-10 The results showed that there was no cross-reaction among the three primer probes, indicating that they had good specificity.

[0193] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A primer set for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus, characterized in that: include: A primer set for detecting the abaA gene of Aspergillus fumigatus, the nucleotide sequence of which is shown in SEQ ID NOs: 1-2; The primer set for detecting the ph1 gene of Aspergillus niger has a nucleotide sequence as shown in SEQ ID NOs: 25-26; and the primer set for detecting the taa gene of Aspergillus flavus has a nucleotide sequence as shown in SEQ ID NOs: 43-44.

2. A probe for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus, characterized in that: include: The nucleotide sequence of the probe for detecting the abaA gene of Aspergillus fumigatus is shown in SEQ ID NO:3; the nucleotide sequence of the probe for detecting the ph1 gene of Aspergillus niger is shown in SEQ ID NO:27; and the nucleotide sequence of the probe for detecting the taa gene of Aspergillus flavus is shown in SEQ ID NO:

45.

3. The probe according to claim 2, characterized in that The 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

4. Use of the primer set according to claim 1 and the probe according to claim 2 or 3 in preparing a kit for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus.

5. A kit for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus, characterized in that: Contains the primer set according to claim 1 and the probe according to claim 2 or 3.

6. The kit according to claim 5, characterized in that The kit also contains a multiplex PCR reaction solution.

7. The kit according to claim 6, characterized in that The multiplex PCR reaction solution includes PCR buffer, DNA polymerase and dNTPs.

8. The kit according to claim 5, characterized in that The kit also contains primers and probes for detecting internal reference genes, the nucleotide sequences of the primers are shown in SEQ ID NOs:46-47, and the nucleotide sequence of the probe is shown in SEQ ID NO:

48.

9. A method for detecting Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus for non-disease diagnosis purposes, characterized in that: Using the DNA of the sample to be tested as a template, PCR is performed using the primer set of claim 1 and the probe of claim 2 or 3, wherein the probes are respectively labeled with different fluorescent markers. When only the fluorescence channel corresponding to the probe detecting the abaA gene of Aspergillus fumigatus has an obvious S-shaped amplification curve and the Ct value is ≤36, the sample to be tested is judged to be positive for Aspergillus fumigatus, and negative for Aspergillus niger and Aspergillus flavus; When only the fluorescence channel corresponding to the probe detecting the ph1 gene of Aspergillus niger shows an obvious S-shaped amplification curve and the Ct value is ≤36, the sample to be tested is judged to be positive for Aspergillus niger, and negative for Aspergillus fumigatus and Aspergillus flavus; When an obvious S-type amplification curve appears only in the fluorescence channel corresponding to the probe detecting the Aspergillus flavus taa gene and the Ct value is ≤35, the sample to be tested is judged to be positive for Aspergillus flavus and negative for Aspergillus fumigatus and Aspergillus niger.

10. The detection method according to claim 9, characterized in that: The PCR reaction system is: 4 μL buffer, 1 μL DNA enzyme, 0.4 μL dNTP, 7.6-11.6 μL water, 2 μL of a mixture of the primer set according to claim 1 and the probe according to claim 2 or 3, 5 μL template, and a total volume of 20 μL; the reaction procedure is: 95°C for 3 minutes, one cycle; 95°C for 5-15 seconds, 56-60°C for 30 seconds, and 40-45 cycles; the volume ratio of the primer-probe mixture of Aspergillus fumigatus, the primer-probe mixture of Aspergillus niger, and the primer-probe mixture of Aspergillus flavus in the mixture is 1:(0.25-1):(0.25-1), and the volume ratio of the primer to the probe in the primer-probe mixture is 2:(0.25-1).

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