Primer probe combination capable of qualitatively or quantitatively detecting four aspergillus simultaneously and application of primer probe combination

Abstract

The invention relates to the technical field of molecular detection of pathogenic microorganisms, and particularly provides a primer probe combination capable of qualitatively or quantitatively detecting four aspergillus at the same time and application of the primer probe combination. The primer probe combination comprises specific primer pairs and probes respectively aiming at aspergillus fumigatus, aspergillus flavus, aspergillus niger and aspergillus terreus. According to the invention, two efficient detection paths are established: rapid qualitative screening of'double-tube quadruple 'can be realized through a multiple fluorescent PCR method, and accurate quantitative analysis of'single-chip quadruple' can also be realized through a multiple quantitative digital PCR method. The verification result shows that aiming at four aspergillus targets, the detection limit of the multiple fluorescent PCR method can reach 75 copies / mL, and the detection limit of the multiple quantitative digital PCR method can reach 300 copies / mL. The scheme has high throughput, high sensitivity and high specificity, and an efficient integrated molecular detection solution is provided for early diagnosis and identification of invasive aspergillosis.

Description

Technical Field

[0001] This invention belongs to the field of Aspergillus detection technology, specifically relating to a primer-probe combination capable of simultaneously qualitatively or quantitatively detecting four types of Aspergillus and its application. Background Technology

[0002] Aspergillus is a filamentous fungus commonly found in moist soil or environments. Its spores can enter the body through respiration and cause disease. Common pathogens include *Aspergillus fumigatus*, *Aspergillus niger*, *Aspergillus flavus*, and *Aspergillus terreus*. Infection is more likely when the body's immune function is suppressed or impaired. Invasive aspergillosis is an opportunistic fungal infection, often co-infecting patients with organ transplants, neutropenia, tumors, immunosuppressants, chronic lung diseases, AIDS, and diabetes. Approximately 20% of patients with invasive pulmonary aspergillosis have no underlying diseases. Long-term overuse of broad-spectrum antibiotics, hormones, immunosuppressants, or weakened immunity caused by severe diseases such as chronic obstructive pulmonary disease, catheterization, peritoneal dialysis, bone marrow transplantation, solid organ transplantation, leukemia, chemotherapy and radiotherapy for tumors, AIDS, and late-stage diabetes can all increase the incidence of deep aspergillosis infections. Deep aspergillosis has a poor prognosis and high mortality rate. Invasive fungal infections have become a major public health problem that seriously affects people's quality of life and threatens their health.

[0003] Traditional methods for detecting deep fungal infections include microscopic morphology, culture, serological immunology, and histopathological morphology. Morphological methods include direct microscopic examination of fungi, culture, and histopathological sections. Microscopic examination has low sensitivity and is prone to missed detections; culture has a long cycle and low sensitivity and positive rate, and species identification requires experience; serological immunology methods detect fungal cell wall antigens in serum, such as the G test and GM test, but have many false positives and false negatives; histopathological morphology involves invasive sampling, and although it can provide direct evidence of fungal invasion, it is not widely used and makes species identification difficult, which is crucial for the treatment of fungal diseases. Fluorescent PCR, with its high specificity and sensitivity, is widely used in the field of microbial detection. Digital PCR, building on the sensitivity and specificity of fluorescent PCR, is beginning to show its advantages with precise quantification. In recent years, resistance of *Aspergillus fumigatus* to triazole drugs has attracted much attention, and *Aspergillus terreus* is naturally resistant to amphotericin B. Clinically, species identification is often necessary for more precise treatment. Therefore, efficient and accurate laboratory detection methods are urgently needed for the etiology of aspergillosis.

[0004] In recent years, multiplex real-time PCR (qPCR) and multiplex digital PCR (dPCR) technologies have demonstrated significant advantages in the field of pathogen detection due to their high specificity, high sensitivity, and quantitative capabilities. Multiplex real-time PCR can simultaneously detect multiple target genes through multi-color fluorescent channels, significantly improving detection efficiency. Quantitative multiplex digital PCR, through droplet or microwell splitting and absolute quantification, overcomes the dependence on standard curves in quantitative real-time PCR, allowing for direct and accurate quantification. It is suitable not only for detecting nucleic acid samples with abundant bacterial loads but also for detecting nucleic acid samples with low bacterial loads. However, existing Aspergillus nucleic acid detection kits generally target single species or rely on single technologies, lacking a solution that integrates the advantages of typing multiplex real-time PCR and multiplex quantitative digital PCR.

[0005] Therefore, developing a nucleic acid detection kit for Aspergillus fungi based on the combination of Aspergillus typing multiplex fluorescent PCR and multiplex quantitative digital PCR to achieve rapid, accurate, quantitative, and multi-target parallel detection is of great practical value for accurately improving the early detection rate of Aspergillus infection and providing laboratory evidence for antifungal treatment. Summary of the Invention

[0006] In view of this, the present invention aims to solve the problems of insufficient sensitivity and specificity, lack of accurate quantification of Aspergillus, poor stability of quality control products, limited detection throughput, and high false positive and false negative rates in the prior art.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a primer-probe combination capable of simultaneously qualitatively or quantitatively detecting four Aspergillus species, including the following primer-probe combination: The primer-probe combination for detecting Aspergillus fumigatus includes primer pairs with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2 and probes with nucleotide sequences as shown in SEQ ID NO.3; The primer-probe combination for detecting Aspergillus flavus includes primer pairs with nucleotide sequences as shown in SEQ ID NO.4 and SEQ ID NO.5 and probes with nucleotide sequences as shown in SEQ ID NO.6; The primer-probe combination for detecting Aspergillus niger includes primer pairs with nucleotide sequences as shown in SEQ ID NO.7 and SEQ ID NO.8 and probes with nucleotide sequences as shown in SEQ ID NO.9; The primer-probe combination for detecting Aspergillus terreus includes primer pairs with nucleotide sequences as shown in SEQ ID NO.10 and SEQ ID NO.11 and probes with nucleotide sequences as shown in SEQ ID NO.12.

[0008] Preferably, the probes for detecting Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus are modified with fluorescent groups and quenching groups at both ends, respectively; the fluorescent groups include any one of FAM, ROX, VIC, HEX, CY5, CY5.5, or Quasar705; the quenching groups include any one of BHQ1, BHQ2, BHQ3, or MGB; when detecting multiple Aspergillus species simultaneously, the fluorescent groups modified at both ends of the probes for different Aspergillus species are different.

[0009] This invention also provides the application of the above-mentioned primer-probe combination in the preparation of multiplex fluorescent PCR detection and / or multiplex quantitative digital PCR detection products.

[0010] The present invention also provides a multiplex fluorescent PCR detection kit capable of simultaneously qualitatively detecting four Aspergillus species, comprising the following components: a detection mixture containing the above-mentioned primer-probe combination, an amplification reaction solution, a negative control, and a positive control.

[0011] Preferably, the detection mixture includes detection mixture 1 and detection mixture 2. Detection mixture 1 includes the primer-probe combination for detecting *Aspergillus fumigatus*, the primer-probe combination for detecting *Aspergillus flavus*, the primer-probe combination for detecting *Aspergillus niger*, and the primer-probe combination for detecting the internal reference GAPDH. Detection mixture 2 includes the primer-probe combination for detecting *Aspergillus terreus* and the primer-probe combination for detecting the internal reference. The primer-probe combination for detecting the internal reference includes primer pairs with nucleotide sequences as shown in SEQ ID NO. 13 and SEQ ID NO. 14 and probes with nucleotide sequences as shown in SEQ ID NO. 15. The final concentration of each primer in detection mixture 1 is 0.2 μM, and the final concentration of each probe is 0.15 μM. The final concentration of each primer in detection mixture 2 is 0.2 μM, and the final concentration of each probe is 0.15 μM.

[0012] The present invention also provides a multiplex quantitative digital PCR detection kit capable of simultaneously quantifying four Aspergillus species, comprising the following components: a detection mixture containing the above-mentioned primer-probe combination, an enzyme, a buffer, a negative control, a positive control, and nuclease-free water.

[0013] Preferably, the negative control is a recombinant plasmid containing a nucleotide sequence as shown in SEQ ID NO. 30, and the positive control is a mixture containing Aspergillus fumigatus recombinant plasmid, Aspergillus flavus recombinant plasmid, Aspergillus niger recombinant plasmid, Aspergillus terreus recombinant plasmid, and an internal reference recombinant plasmid; the Aspergillus fumigatus recombinant plasmid contains the Aspergillus fumigatus fragment with a nucleotide sequence as shown in SEQ ID NO. 18, the Aspergillus flavus recombinant plasmid contains the Aspergillus flavus fragment with a nucleotide sequence as shown in SEQ ID NO. 21, the Aspergillus niger recombinant plasmid contains the Aspergillus niger fragment with a nucleotide sequence as shown in SEQ ID NO. 24, the Aspergillus terreus recombinant plasmid contains the Aspergillus terreus fragment with a nucleotide sequence as shown in SEQ ID NO. 27, and the internal reference recombinant plasmid contains the internal reference fragment with a nucleotide sequence as shown in SEQ ID NO. 30.

[0014] Preferably, the negative and positive controls contain a DNA protectant, said DNA protectant including salmon sperm DNA and DNA nuclease inhibitors.

[0015] Preferably, in the multiplex fluorescent PCR detection kit, the amplification reaction program is 37℃ for 5 min; 95℃ for 5 min; 95℃ for 15 s and 60℃ for 30 s, for 45 cycles. The result determination method is as follows: the negative control and positive control should simultaneously meet the following conditions, otherwise the test is considered invalid and needs to be repeated: if the negative control Ct value is >40 or "Undetermined", and the internal reference Ct value is <44, and the positive control Ct value is ≤40, and the internal reference Ct value is <44; if the test is valid, the test sample is determined: if the sample Ct value is ≤40, the internal reference Ct value is <44 or no internal reference Ct value is detected, it indicates a positive result; if the sample Ct value is >40 or "Undetermined", and the internal reference Ct value is <44, it indicates a negative result.

[0016] Preferably, in the multiplex quantitative digital PCR detection kit, the final concentration of each primer in the detection mixture is 0.2 μM, and the final concentration of each probe is 0.15 μM; the amplification reaction program is: oil curing at 50℃ for 5 min; pre-denaturation at 95℃ for 5 min; cycling reaction at 95℃ for 15 s and 60℃ for 30 s, for 45 cycles; holding at 25℃; the result determination method is as follows: the negative control and positive control should simultaneously meet the following conditions, otherwise the detection is considered invalid and needs to be repeated: the one-dimensional scatter plot shows that the positive control is clearly divided into upper and lower layers, the negative control signal points are concentrated in the lower layer of the positive control with consistent fluorescence intensity, and there are no discrete droplets compared with the upper part of the positive control; if the detection is valid, the sample to be tested is determined: the number of positive points of the internal reference is <3, indicating that the quantitative result is invalid; the number of positive points of the sample is <1, and the number of positive points of the internal reference is ≥3, indicating that the result is negative; the number of positive points of the sample is ≥1, and the number of positive points of the internal reference is ≥3, indicating that the result is positive, and the quantitative value is output.

[0017] The beneficial effects of this invention are: The primer-probe combination provided by this invention enables qualitative detection using dual-tube quadruple assays via multiplex fluorescent PCR, and quantitative detection using single-chip quadruple assays via multiplex quantitative digital PCR. When using the primer-probe combination provided by this invention for multiplex fluorescent PCR detection of four Aspergillus targets (Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus), the detection limit for all four targets is 75 copies / mL. When using the primer-probe combination provided by this invention for multiplex quantitative digital PCR detection of the same four targets, the detection limit for all four targets is 300 copies / mL (0.3 copies / μL).

[0018] The multiplex fluorescent PCR detection kit and multiplex quantitative digital PCR detection kit provided by this invention can specifically and sensitively detect Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus, and are suitable for early typing and qualitative detection as well as accurate quantitative detection of suspected Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus infections in clinical practice. Attached Figure Description

[0019] Figure 1 The amplification curves of positive controls for Aspergillus fumigatus (FAM), Aspergillus flavus (ROX), Aspergillus niger (VIC), and internal control (CY5) were obtained by multiplex fluorescent PCR. Figure 2 The amplification curves of Aspergillus terreus (FAM) and the internal control (CY5) were obtained by multiplex fluorescent PCR. Figure 3 One-dimensional scatter plots were generated for the detection of Aspergillus fumigatus (FAM), Aspergillus flavus (HEX), Aspergillus niger (ROX), Aspergillus terreus (CY5), and internal control (QUASAR705) by multiplex quantitative digital PCR. Detailed Implementation

[0020] This invention provides a primer-probe combination capable of simultaneously qualitatively or quantitatively detecting four Aspergillus species, including the following primer-probe combination: The primer-probe combination for detecting Aspergillus fumigatus includes primer pairs with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2 and probes with nucleotide sequences as shown in SEQ ID NO.3; The primer-probe combination for detecting Aspergillus flavus includes primer pairs with nucleotide sequences as shown in SEQ ID NO.4 and SEQ ID NO.5 and probes with nucleotide sequences as shown in SEQ ID NO.6; The primer-probe combination for detecting Aspergillus niger includes primer pairs with nucleotide sequences as shown in SEQ ID NO.7 and SEQ ID NO.8 and probes with nucleotide sequences as shown in SEQ ID NO.9; The primer-probe combination for detecting Aspergillus terreus includes primer pairs with nucleotide sequences as shown in SEQ ID NO.10 and SEQ ID NO.11 and probes with nucleotide sequences as shown in SEQ ID NO.12.

[0021] The primer-probe combination provided by this invention has the following advantages: (1) It can specifically detect four common Aspergillus species: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus. (2) It has high specificity: it has no cross-reactivity with common clinical fungi (such as Candida spp., Mucorales, Penicillium spp., Cryptococcus spp., Fusarium spp., Pneumocystis jirovecii, Blue styloides marneffei, etc.) and bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, etc.). (3) It has high sensitivity: when performing multiplex fluorescent PCR detection, it can still be detected when the target gene content is 75 copies / mL; when performing multiplex quantitative digital PCR detection, the detection sensitivity is 300 copies / mL (0.3 copies / μL).

[0022] (4) Short processing time: DNA can be directly extracted from the sample for detection. Multiplex fluorescent PCR takes 1.5 hours, and multiplex quantitative digital PCR takes 2.0 hours. (5) When performing multiplex quantitative digital PCR, the target can be directly and absolutely quantified without the need to establish a standard curve. The method is simple. (6) When performing multiplex quantitative digital PCR, the results are clear and easy to interpret, and the distinction between positive and negative results is obvious.

[0023] In this invention, the probes for detecting Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus are preferably modified with fluorescent and quenching groups at both ends, respectively. The fluorescent groups preferably include any one of FAM, ROX, VIC, HEX, CY5, CY5.5, or Quasar705. The quenching groups preferably include any one of BHQ1, BHQ2, BHQ3, or MGB. When detecting multiple Aspergillus species simultaneously, the fluorescent groups modified at both ends of the probes for different Aspergillus species are different. In a specific embodiment of the present invention, when performing multiplex fluorescent PCR, it is preferred to place *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus niger*, and an internal control in one tube, and *Aspergillus terreus* and the internal control in another tube. Both tubes are then simultaneously placed on a fluorescent PCR instrument for detection. The probe for *Aspergillus fumigatus* is modified with a FAM fluorescent group, the probe for *Aspergillus flavus* is modified with a ROX fluorescent group, the probe for *Aspergillus niger* is modified with a VIC fluorescent group, and the probe for the internal control is modified with a CY5 fluorescent group. In the other tube, the probe for *Aspergillus terreus* is modified with a FAM fluorescent group, and the probe for the internal control is modified with a CY5 fluorescent group. In a specific embodiment of the present invention, when performing multiplex quantitative digital PCR, the probe for *Aspergillus fumigatus* is modified with a FAM fluorescent group, the probe for *Aspergillus flavus* is modified with a VIC or HEX fluorescent group, the probe for *Aspergillus niger* is modified with a ROX fluorescent group, the probe for *Aspergillus terreus* is modified with a CY5 fluorescent group, and the probe for the internal control is modified with a QUASAR705 or CY5.5 fluorescent group.

[0024] This invention also provides the application of the above-mentioned primer-probe combination in the preparation of multiplex fluorescent PCR detection and / or multiplex quantitative digital PCR detection products.

[0025] The present invention also provides a multiplex fluorescent PCR detection kit capable of simultaneously qualitatively detecting four Aspergillus species, comprising the following components: a detection mixture containing the above-mentioned primer-probe combination, an amplification reaction solution, a negative control, and a positive control.

[0026] The detection principle of this invention's multiplex fluorescent PCR detection kit is as follows: Multiple primer pairs are designed based on specific conserved fragments of *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus niger*, and *Aspergillus terreus*. The presence of specific PCR products is determined by detecting the intensity of the fluorescence signal and the shape of the amplification curve. Based on analysis of different fluorescence channels, *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus niger*, and / or *Aspergillus terreus* can be rapidly identified, thereby determining whether the tested sample contains the target DNA fragment and, if so, which type of target DNA fragment. This invention's multiplex fluorescent PCR kit is used for the qualitative, rapid, and efficient detection of nucleic acid fragments from *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus niger*, and / or *Aspergillus terreus*.

[0027] Given that most commercially available fluorescence PCR instruments use four-channel configurations, the detection mixture in this invention's multiplex fluorescence PCR detection kit is divided into detection mixture 1 and detection mixture 2. Detection mixture 1 preferably includes primer-probe combinations for detecting *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus niger*, and the internal control GAPDH. The probes in these four primer-probe combinations are modified with four different fluorescent groups: FAM, ROX, VIC, and CY5. *Aspergillus fumigatus*, *Aspergillus niger*, *Aspergillus flavus*, and the internal control each occupy one channel. In detection mixture 2, *Aspergillus terreus* and the internal control each occupy one channel. Detection mixture 1 and detection mixture 2 are prepared separately, but can be used together in the same fluorescence PCR instrument for detection, thus achieving simultaneous qualitative detection of four *Aspergillus* targets. The primer-probe combination for detecting the internal control preferably includes primer pairs with nucleotide sequences as shown in SEQ ID NO. 13 and SEQ ID NO. 14 and probes with nucleotide sequences as shown in SEQ ID NO. 15. In the multiplex fluorescence PCR detection kit of the present invention, the final concentration of each primer in detection mixture 1 is 0.2 μM, and the final concentration of each probe is 0.15 μM; the final concentration of each primer in detection mixture 2 is 0.2 μM, and the final concentration of each probe is 0.15 μM. In the multiplex fluorescence PCR detection kit of the present invention, the preferred amplification reaction solution is Hieff Unicon® 2×MIX (Hieff Unicon® Multiplex Quantitative PCR Reaction Premix), purchased from Hieff Biotechnology (Shanghai) Co., Ltd., catalog number 11212ES03.

[0028] The present invention also provides a multiplex quantitative digital PCR detection kit capable of simultaneously quantifying four Aspergillus species, comprising the following components: a detection mixture containing the above-mentioned primer-probe combination, an enzyme, a buffer, a negative control, a positive control, and nuclease-free water.

[0029] This invention's multiplex quantitative digital PCR detection kit employs a chip-based digital PCR method. The copy number of the target nucleic acid molecule is calculated based on the ratio of negative to positive droplets after the reaction. This method achieves nucleic acid sample separation during the PCR reaction, reduces background signal, and significantly improves the detection sensitivity for low-copy samples. This multiplex quantitative digital PCR detection kit is used for the rapid and efficient quantitative detection of nucleic acid fragments from *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus niger*, and *Aspergillus terreus*. In this multiplex quantitative digital PCR detection kit, the preferred Taq enzyme is BioDigital Maxuseful dPCR Kit (U+), and the preferred buffer is 3×dPCR Buffer: 3×Maxuseful dPCR Buffer. Both the enzyme and buffer were purchased from Shanghai Xiaohaigui Technology Co., Ltd. Catalog number: C02301. Both kits provided by this invention contain a low-concentration quality control system, including a positive control containing a mixed plasmid of target sequences from *Aspergillus fumigatus*, *Aspergillus niger*, *Aspergillus flavus*, and *Aspergillus terreus*, and an internal control, at a concentration of 80-120 copies / μL, to ensure correct experimental procedures and normal operation of reagents and instruments; and a negative control containing an internal control plasmid, at a concentration of 80-120 copies / μL, to monitor for experimental contamination. A DNA protectant is preferably added to the storage matrix of the quality control system; the preferred DNA protectant includes salmon sperm DNA and a DNA nuclease inhibitor; the preferred storage temperature for the quality control is -20℃ ± 10℃. In this invention, the negative control is preferably a recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO. 30, and the positive control is preferably a mixture containing Aspergillus fumigatus recombinant plasmid, Aspergillus flavus recombinant plasmid, Aspergillus niger recombinant plasmid, Aspergillus terreus recombinant plasmid, and an internal reference recombinant plasmid. The Aspergillus fumigatus recombinant plasmid preferably contains the Aspergillus fumigatus fragment with the nucleotide sequence shown in SEQ ID NO. 18; the Aspergillus flavus recombinant plasmid preferably contains the Aspergillus flavus fragment with the nucleotide sequence shown in SEQ ID NO. 21; the Aspergillus niger recombinant plasmid preferably contains the Aspergillus niger fragment with the nucleotide sequence shown in SEQ ID NO. 24; the Aspergillus terreus recombinant plasmid preferably contains the Aspergillus terreus fragment with the nucleotide sequence shown in SEQ ID NO. 27; and the internal reference recombinant plasmid contains the internal reference fragment with the nucleotide sequence shown in SEQ ID NO. 30. The final concentration of each recombinant plasmid in the multiplex quantitative digital PCR kit is preferably 100 ± 20 copies / μL.

[0030] In the multiplex fluorescent PCR detection kit of this invention, the amplification reaction program is 37℃ for 5 min; 95℃ for 5 min; 95℃ for 15 s and 60℃ for 30 s, for 45 cycles. The result determination method is as follows: the negative control and positive control should simultaneously meet the following conditions, otherwise the test is considered invalid and needs to be repeated: if the negative control Ct value is >40 or "Undetermined", and the internal reference Ct value is <44, and the positive control Ct value is ≤40, and the internal reference Ct value is <44; if the test is valid, the test sample is determined: if the sample Ct value is ≤40, the internal reference Ct value is <44 or no internal reference Ct value is detected, it indicates a positive result; if the sample Ct value is >40 or "Undetermined", and the internal reference Ct value is <44, it indicates a negative result.

[0031] In the multiplex quantitative digital PCR detection kit of this invention, the final concentration of each primer in the detection mixture is 0.2 μM, and the final concentration of each probe is 0.15 μM. The amplification reaction program is as follows: oil curing at 50℃ for 5 min; pre-denaturation at 95℃ for 5 min; cycling reaction at 95℃ for 15 s and 60℃ for 30 s, for 45 cycles; incubation at 25℃. The result determination method is as follows: the negative control and positive control should simultaneously meet the following conditions, otherwise the detection is considered invalid and needs to be repeated: the one-dimensional scatter plot shows that the positive control is clearly divided into upper and lower layers, the negative control signal points are concentrated in the lower layer of the positive control with consistent fluorescence intensity, and there are no discrete droplets compared with the upper part of the positive control. If the detection is valid, the sample to be tested is determined: if the number of positive points of the internal reference is <3, the quantitative result is invalid; if the number of positive points of the sample is <1 and the number of positive points of the internal reference is ≥3, the result is negative; if the number of positive points of the sample is ≥1 and the number of positive points of the internal reference is ≥3, the result is positive, and the quantitative value is output.

[0032] The present invention also provides a method for detecting whether a sample contains four types of Aspergillus, preferably comprising the following steps: First, DNA was extracted from the test samples, and an internal reference gene primer-probe set was added to verify the extraction efficiency. Then, a multiplex fluorescent PCR detection kit was used for typing and qualitative analysis. Positive samples were further quantified using a multiplex quantitative digital PCR detection kit. Finally, the multiplex quantitative digital PCR detection data were verified by a quality control system, and the copy number detection results for the four Aspergillus target genes were output. If the Ct value detected by the multiplex fluorescent PCR method was ≤40, it was considered a preliminary positive and required confirmation by multiplex quantitative digital PCR. If the multiplex quantitative digital PCR result was inconsistent with the multiplex fluorescent PCR result, the multiplex quantitative digital PCR result shall prevail.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Unless otherwise specified, the following embodiments are all conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] Example 1 Primer and probe screening 1. Primer specificity testing Streptococcus pneumoniae (BNCC338425, sold by Beina Chuanglian Biotechnology Co., Ltd.), Pseudomonas aeruginosa (CMCC(B)10104, sold by Nanjing Lezhen Biotechnology Co., Ltd.), Enterococcus faecalis (BNCC102668, sold by Beina Chuanglian Biotechnology Co., Ltd.), Staphylococcus aureus (MCC(CB)26003, sold by Nanjing Lezhen Biotechnology Co., Ltd.), Staphylococcus epidermidis (BNCC330867, sold by Beina Chuanglian Biotechnology Co., Ltd.), and Klebsiella pneumoniae (CNS) were collected separately. The following bacteria are listed: * **Bacterium faecium (ATCC(B)46117)** (from Nanjing Lezhen Biotechnology Co., Ltd., catalog number BNCC324384), * **Escherichia coli (from Nanjing Lezhen Biotechnology Co., Ltd., catalog number CMCC(B)44102)**, * **Proteus mirabilis (from Nanjing Lezhen Biotechnology Co., Ltd., catalog number ATCC35659)**, * **Nocardia asteroides (from Nanjing Lezhen Biotechnology Co., Ltd., catalog number BNCC152914)**, and * **Moraxella catarrhalis (from Nanjing Lezhen Biotechnology Co., Ltd., catalog number BNCC275822)**. Haemophilus influenzae (BNCC359479, Beijing Beina Chuanglian Biotechnology Co., Ltd.), Mycobacterium marinum (BNCC360708, Beijing Beina Chuanglian Biotechnology Co., Ltd.), Mycobacterium abscessus (Bio-115238, Beijing Bio-115238, Beijing Mycobacterium avium (CCUG27851, Beijing Zhongke Quality Inspection Biotechnology Co., Ltd.), Mycobacterium intracellularis (CCUG28005T, Beijing Zhongke Quality Inspection Biotechnology Co., Ltd.), Mycobacterium bovis (CCUG37460, Beijing Zhongke Quality Inspection Biotechnology Co., Ltd.), Mycobacterium kansasii (CCUG27787, Beijing Zhongke Quality Inspection Biotechnology Co., Ltd.), Mycobacterium tuberculosis (inactivated) (BNCC263834, Beijing Beina Chuanglian Biotechnology Co., Ltd.), Scrofula Mycobacterium spp. (BNCC364272, Shanghai Beina Chuanglian Biotechnology Co., Ltd.), Candida albicans (CMCC(F)98001, Nanjing Lezhen Biotechnology Co., Ltd.), Candida tropicalis (ATCC(F)750, Nanjing Lezhen Biotechnology Co., Ltd.), Candida glabrata (BNCC337348, Shanghai Beina Chuanglian Biotechnology Co., Ltd.), Candida parapsilosis (BNCC378088, Shanghai Beina Chuanglian Biotechnology Co., Ltd.), Candida krusei (ATCC6258, Nanjing Lezhen Biotechnology Co., Ltd.), Saccharomyces cerevisiae (336054, Shanghai Beina Chuanglian Biotechnology Co., Ltd.), Rhizopus umbellatus (356134, Shanghai Beina Chuanglian Biotechnology Co., Ltd.), Rhizopus oligosporus (YS-MCC3, Shanghai Yansheng Industrial Co., Ltd.).178), *Braziliana spp.* (Beijing Bio-Biotechnology Co., Ltd., bio-108911), *Rhizopus microsporum* (Shangcheng Beina Chuanglian Biotechnology Co., Ltd., 145129), *Mucor racemosa* (Shangcheng Beina Chuanglian Biotechnology Co., Ltd., 189210), *Millettia microsporum* (Beijing Bio-Biotechnology Co., Ltd., bio-84574), *Aspergillus variegata* (Shangcheng Beina Chuanglian Biotechnology Co., Ltd., BNCC146413), *Aspergillus nidus* (Shangcheng Beina Chuanglian Biotechnology Co., Ltd., BNCC185653) * *Cyperus spicata* (BNCC374366, Beina Chuanglian Biotechnology Co., Ltd.), *Fusarium oxysporum* (BNCC120618, Beina Chuanglian Biotechnology Co., Ltd.), *Aspergillus oryzae* (BNCC338380, Beina Chuanglian Biotechnology Co., Ltd.), *Aspergillus bleachingii* (BNCC357832, Beina Chuanglian Biotechnology Co., Ltd.), *Aspergillus polyplosporum* (BNCC336544, Beina Chuanglian Biotechnology Co., Ltd.), *Fusarium solani* (BNCC12154, Beina Chuanglian Biotechnology Co., Ltd.) 7) *Penicillium citrinum* (BNCC336236, Beina Chuanglian Biotechnology Co., Ltd.), *Aspergillus glaucus* (BNCC359583, Beina Chuanglian Biotechnology Co., Ltd.), Human genomic DNA (BNCC372385, Beina Chuanglian Biotechnology Co., Ltd.), *Cryptococcus neoformans* (BNCC362607, Beina Chuanglian Biotechnology Co., Ltd.), *Cryptococcus grease* (354892, Beina Chuanglian Biotechnology Co., Ltd.), *Cryptococcus lorentzum* (189135, Beina Chuanglian Biotechnology Co., Ltd.). Genomic DNA from *Aspergillus fumigatus* (BNCC338385), *Aspergillus flavus* (BNCC336156), *Aspergillus niger* (CMCC(F)98003), and *Aspergillus terreus* (BNCC335941) was collected. Specificity tests were performed using different primers. As shown in Table 1, the final confirmed primers and probes exhibited good specificity and did not cross-react with common human and clinical bacteria and fungi. The final confirmed primer and probe sequences are shown in Table 2.

[0037] Table 1. Experimental results of primer specificity test

[0038] Note: "NoCt" means "No Ct value detected". Table 2 Confirmed Primer-Probe Sequences

[0039] Example 2 Preparation of plasmids for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and internal reference plasmids (construction of cloning plasmids). 1. Genomic DNA of *Aspergillus fumigatus* was obtained. Using this genomic DNA as a template, PCR amplification was performed using primers SEQ ID NO.16 DL-1: 5'-AGATCATCATCATCTTGGTTATGAG-3' and SEQ ID NO.17 DL-2: 5'-TTTTCCTTTAATTACGGGTTAGAC-3'. The amplified *Aspergillus fumigatus* positive control DNA fragment (nucleotide sequence shown in SEQ ID NO.18, 903 bp) was obtained. The PCR product was recovered and purified, and then inserted into the plasmid vector pUC57 with the EcoRI restriction site at the multiple cloning site (MCS). This was transformed into *E. coli* DH5α, and the recombinant transformants were sequenced for verification. After culturing the recombinant transformants, the plasmid was extracted to obtain the *Aspergillus fumigatus* plasmid. The entire process was handled by Shanghai Sangon Biotech Co., Ltd.

[0040] 2. Genomic DNA of *Aspergillus flavus* was obtained. Using this genomic DNA as a template, PCR amplification was performed using primers SEQ ID NO.19 DL-3: 5'-TTCAAGAACAGCCTGCACAGAG-3' and SEQ ID NO.20 DL-4: 5'-ACTACCCAGGAAAGGGATCCAG-3'. The amplified *Aspergillus flavus* DNA fragment (nucleotide sequence shown in SEQ ID NO.21, 837 bp) was obtained. The PCR product was recovered and purified, ligated into the plasmid vector pUC57 containing the EcoRI restriction site of MCS, transformed into *E. coli* DH5α, and the recombinant transformants were sequenced for verification. After culturing the recombinant transformants, the plasmid was extracted to obtain the *Aspergillus flavus* plasmid. This process was handled by Shanghai Sangon Biotech Co., Ltd.

[0041] 3. Genomic DNA of *Aspergillus niger* was obtained. Using this genomic DNA as a template, PCR amplification was performed using primers SEQ ID NO.22 DL-5: 5'-TCCGCGGGCGTTGCTCTAT-3' and SEQ ID NO.23 DL-6: 5'-AGATGGGGACAACGTGGGTGA-3'. The amplified *Aspergillus niger* DNA fragment (nucleotide sequence as shown in SEQ ID NO.24, 835 bp) was obtained. The PCR product was recovered and purified, ligated into the plasmid vector pUC57 containing the EcoRI restriction site of MCS, transformed into *E. coli* DH5α, and the recombinant transformants were sequenced for verification. After culturing the recombinant transformants, the plasmid was extracted to obtain the *Aspergillus niger* plasmid. This process was handled by Shanghai Sangon Biotech Co., Ltd.

[0042] 4. Genomic DNA of *Aspergillus terreus* was obtained. Using this genomic DNA as a template, PCR amplification was performed using primers SEQ ID NO.25 DL-7: 5'-AGACAGGTCTCCGAATACAAGG-3' and SEQ ID NO.26 DL-8: 5'-AAACTCGTTGTCTAGAAGGCAGTC-3'. The amplified *Aspergillus terreus* DNA fragment (nucleotide sequence shown in SEQ ID NO.27, 732 bp) was obtained. The PCR product was recovered and purified, ligated into the plasmid vector pUC57 containing the EcoRI restriction site of MCS, transformed into *E. coli* DH5α, and the recombinant transformants were sequenced for verification. After culturing the recombinant transformants, the plasmid was extracted to obtain the *Aspergillus terreus* plasmid. This process was handled by Shanghai Sangon Biotech Co., Ltd.

[0043] 5. The construction process of the internal control plasmid is as follows: Human genomic DNA was obtained. Using human genomic DNA as a template, PCR amplification was performed using primer A: SEQ ID NO.28: 5'-TGCAAGGCGATTAAGTTGGGTA-3' and primer B: SEQ ID NO.29: 5'-TGCGGTGGGAGATCTGGTTATTA-3' to obtain a human DNA fragment (nucleotide sequence as shown in SEQ ID NO.30, 1077bp). This human DNA fragment was ligated into the plasmid vector pUC57 containing the EcoRI restriction site of MCS to construct the internal reference plasmid. This process was specifically completed by the Hangzhou branch of Beijing Qingke Biotechnology Co., Ltd.

[0044] Example 3 Establishment of multiplex fluorescent PCR and multiplex quantitative digital PCR systems Using the Aspergillus fumigatus plasmid, Aspergillus flavus plasmid, Aspergillus niger plasmid, and Aspergillus terreus plasmid obtained in Example 2 as test samples, the following tests were conducted: 1. Annealing temperature selection The annealing temperatures of four primer probes (Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus) were adjusted (58, 60, and 62 °C). Aspergillus fumigatus (FAM), Aspergillus flavus (ROX), Aspergillus niger (VIC), and the internal control CY5 were placed in one tube, while Aspergillus terreus (FAM) and the internal control CY5 were placed in another tube. The results are shown in Table 3, indicating that the sensitivity of the four Aspergillus species was best when the annealing temperature was 60 °C.

[0045] Table 3 Results of adjusting annealing temperature (Ct values)

[0046] 2. Establishment of a multiplex fluorescent PCR system 2.1 Study of the reaction solution Three reaction solutions were selected for amplification using Aspergillus multiplex fluorescence PCR. Aspergillus fumigatus (FAM), Aspergillus flavus (ROX), Aspergillus niger (VIC), and the internal control CY5 were in one tube, while Aspergillus terreus (FAM) and the internal control CY5 were in another tube. The results are shown in Table 4. Reaction solution 2 showed the best performance; the amplification of each target gene and the internal control using reaction solution 2 was more sensitive than the other two reaction solutions. Reaction solution 2 was Hieff Unicon® 2×MIX (Hieff Unicon® Multiplex Quantitative PCR Reaction Premix), purchased from Hieff Biotechnology (Shanghai) Co., Ltd., catalog number 11212ES03.

[0047] Table 4 Results of reaction solution selection (CT value)

[0048] 2.2 Study on the Concentration of Mixed Solution To optimize detection sensitivity, primer and probe concentrations were optimized. The results of probe concentration optimization are shown in Table 5. It is evident that a final probe concentration of 0.15 μM was optimal for both detection mixture 1 and detection mixture 2, resulting in the best sensitivity for each target gene and internal control.

[0049] Table 5 Optimization of probe concentration

[0050] Based on the optimization of probe concentration, primer concentration was further optimized, and the results are shown in Table 6. It can be seen that when the final primer concentration was 0.2 μM in both detection mixture 1 and detection mixture 2, the sensitivity of each target gene and internal control was optimal.

[0051] Table 6 Optimization of primer concentration

[0052] 3. Establishment of a multiplex quantitative digital PCR system 3.1 Study of the reaction solution Five reaction solutions were selected for multiplex quantitative digital PCR amplification of *Aspergillus fumigatus*, *Aspergillus flavus*, *Aspergillus niger*, and *Aspergillus terreus*. The results are shown in Table 7. Reaction solution 5 showed the best performance, with the highest signal-to-noise ratio and highest concentration (copies / mL) for each target gene and internal control. Reaction solution 5 is a general digital PCR kit purchased from Shanghai Xiaohaigui Technology Co., Ltd. Catalog number: C02301 (current catalog number is SJ24).

[0053] Table 7. Study of the reaction solution

[0054] 3.2 Investigation into the concentration of the mixed solution To optimize the performance of multiplex quantitative digital PCR, primer and probe concentrations were optimized. The results are shown in Table 8. It can be seen that the signal-to-noise ratio was highest and the concentration (copies / mL) was highest when the primer-probe concentration was 0.2 μM:0.15 μM for all detection mixtures.

[0055] Table 8 Optimization of primer and probe concentrations

[0056] Example 4 Composition of Multiplex Fluorescent PCR and Multiplex Quantitative Digital PCR Kits 4.1 Composition of the Multiplex Fluorescent PCR Kit (1) Detection mixture 1: containing Aspergillus fumigatus primers and probes (modified with FAM fluorescent group); Aspergillus flavus primers and probes (modified with ROX fluorescent group); Aspergillus niger primers and probes (modified with VIC fluorescent group); internal control primers and probes (modified with CY5 fluorescent group); the final concentration of each primer is 0.2 μM, and the final concentration of each probe is 0.15 μM.

[0057] (2) Detection mixture 2: containing Aspergillus terrestris primers and probes (modified with FAM fluorescent groups); internal reference primers and probes (modified with CY5 fluorescent groups); the final concentration of each primer is 0.2 μM, and the final concentration of each probe is 0.15 μM.

[0058] The specific sequences of each primer and probe in steps (1) and (2) are shown in Table 2.

[0059] (3) Negative control: plasmid containing the human housekeeping gene GAPDH fragment (internal reference plasmid, prepared in Example 2), with a final concentration of 0.1 ng / mL, 0.1×TE buffer, salmon extract and nuclease inhibitor.

[0060] (4) Positive control: A mixture of cloning plasmids containing positive control DNA sequences of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus and internal control plasmids (each plasmid was prepared in Example 2). The final concentrations of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus and internal control plasmids were all 0.1 ng / mL. 0.1×TE buffer, salmon extract and nuclease inhibitors were also included.

[0061] (5) Fluorescent PCR reaction solution: Hieff Unicon® 2×MIX (Hieff Unicon® multiplex fluorescent quantitative PCR reaction premix), containing dUDG (containing dUTP), Taq enzyme, UDG enzyme, MgCl2, buffer, etc., purchased from Hieff Biotechnology Shanghai Co., Ltd., catalog number 11212ES03.

[0062] The dosage of each component in the multiplex fluorescent PCR kit is shown in Table 9.

[0063] Table 9. Dosage of each component in the multiplex fluorescence PCR kit

[0064] When using a multiplex fluorescent PCR kit for detection, the amplification curves for test samples that are positive for all four Aspergillus species are shown in the diagram below. Figure 1 and Figure 2 As shown.

[0065] 4.2 Composition of the Multiplex Quantitative Digital PCR Reagent Kit (1) Detection mixture: containing Aspergillus fumigatus primers and probes (modified with FAM fluorescent group); Aspergillus flavus primers and probes (modified with HEX fluorescent group); Aspergillus niger primers and probes (modified with ROX fluorescent group); Aspergillus terreus primers and probes (modified with CY5 fluorescent group); internal control primers and probes (modified with QUASAR705 fluorescent group); the final concentration of each primer was 0.2 μM, and the final concentration of each probe was 0.15 μM. The specific sequences of each primer and probe are shown in Table 2.

[0066] (2) Negative control: a plasmid containing a fragment of the human housekeeping gene (GAPDH) (internal reference plasmid, prepared in Example 2), with a final concentration of 100±20 copies / μL, 0.1×TE buffer, salmon extract and nuclease inhibitor.

[0067] (3) Positive control: A mixture of cloning plasmids containing positive control DNA sequences of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus and internal control plasmids (each plasmid was prepared in Example 2). The final concentrations of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus and internal control plasmids were 100±20 copies / μL. 0.1×TE buffer, salmon extract and nuclease inhibitors were also included.

[0068] (4) Taq enzyme: Taq DNA polymerase.

[0069] (5) 3×dPCR Buffer: 3×Maxusefμl PCR Buffer.

[0070] Taq enzyme and 3×dPCR Buffer were purchased from Shanghai Little Turtle Technology Co., Ltd., catalog number: C02301 (now catalog number is SJ24).

[0071] (6) Nuclease-free water.

[0072] (7) Consumables (purchased from Shanghai Little Turtle Technology Co., Ltd.): Chip (item number HC11), BioDigital digital PCR oil (item number: HC31, which includes oil phase A and oil phase B, and its specific function is to seal the gaps in the chip except for the reaction system after mixing oil phase A and oil phase B), auxiliary materials (item number: HC21, which includes pipette tips, eight-tube strips, eight-tube strip caps, oil tray plates, waste boxes, etc.).

[0073] The dosage of each component in the multiplex quantitative digital PCR kit is shown in Table 10.

[0074] Table 10. Dosage of each component in the multiplex quantitative digital PCR kit

[0075] When using a multiplex quantitative digital PCR kit for detection, the amplification curve diagram for a test sample that is positive for all four Aspergillus species is shown below. Figure 3 As shown.

[0076] Example 5 Detection method of multiplex fluorescent PCR Before using the multiplex fluorescent PCR kit described in Example 4.1, DNA needs to be extracted from the sample to be tested. DNA extraction can be performed using the DILAN Bio Nucleic Acid Extraction or Purification Kit, catalog number DL-A-006.

[0077] 1. Preparation of PCR reaction tubes (reagent preparation area) (1) Determine the number of reaction tubes n (number of samples + negative control + positive control); take out test mixture 1, test mixture 2 and amplification reaction solution, and thaw them on ice or at room temperature. All kit components need to be briefly centrifuged before use. Each reaction system is shown in Table 11.

[0078] Table 11 Reaction system for multiplex fluorescence PCR

[0079] Calculate the amount of each reagent (except for the sample / control) according to the number of reaction tubes n, and add them to the centrifuge tubes. Mix thoroughly (it is recommended to use a pipette to slowly and repeatedly mix while avoiding liquid splashing or generating a large number of bubbles). After short-term centrifugation, aliquot 15 μL into each PCR reaction tube.

[0080] 2. Sample addition (sample processing area or sample addition area) Add 10 μL of the DNA of the sample to be tested, or the negative control or positive control sample to the prepared PCR reaction tube, tighten the cap (or attach the sealing film), centrifuge briefly, and transfer to the sample detection area.

[0081] 3. PCR amplification and fluorescence detection (sample detection area) Place the prepared reaction tubes in a fluorescence PCR instrument (ABI 7500) and perform amplification and detection according to the conditions listed in Table 12 based on the edited sample information.

[0082] Table 12 Amplification reaction procedure for multiplex fluorescent PCR

[0083] 4. Setting conditions for result analysis (1) When analyzing the amplification curve results, the plot type can generally be set as: ΔRn vsCycle.

[0084] (2) Baseline setting: The analysis software of the fluorescence PCR instrument can automatically set the baseline, usually from cycle number 2 to the 3 cycles before the first amplification curve.

[0085] (3) Threshold setting: The analysis software of the fluorescence PCR instrument can automatically set the threshold line or set it manually. Usually, the threshold line is set at the part of the exponential growth phase of the amplification curve above the baseline. The point where the amplification curve intersects with the threshold line is the Ct value, which represents the variable value ΔRn of the normalized reporter fluorescence intensity (Rn) before and after amplification [ΔRn = Rn (reading after PCR amplification) - Rn (reading before PCR amplification)]. The Ct value is linearly negatively correlated with the logarithmic amount of the target DNA fragment at the beginning of the reaction.

[0086] 5. Quality Control Standards The negative and positive controls for this kit must simultaneously meet the following conditions; otherwise, the experiment is considered invalid and needs to be repeated: Negative control: negative control Ct value > 40 or "Undetermined", while internal reference Ct value < 44.

[0087] Positive control: positive control Ct value ≤ 40, and internal reference Ct value < 44.

[0088] 6. Reading the experimental results Taking the ABI 7500 as an example, other models should refer to the software manual that comes with that model. According to the analysis software manual that comes with the ABI 7500 instrument, first set the baseline and threshold (it is recommended to select automatic for both baseline and threshold settings), then click "Analyze" in the software. The system will automatically generate the results. Observe the Ct value of the amplification curve for each sample and export it to an Excel file.

[0089] The instrument's software was used for automatic analysis to obtain the Ct values ​​for each sample, and the results were determined according to Table 13.

[0090] Table 13 Method for Determining Ct Values

[0091] In the presence of high concentrations of the target gene, its amplification may lead to a negative result in the internal control test.

[0092] If the internal control channel (CY5) shows S-type amplification and meets the following conditions (Table 14), it is considered positive: Table 14. Result Judgment Methods for Each Channel

[0093] Example 6 Detection method of multiplex quantitative digital PCR Before using the multiplex quantitative digital PCR kit described in Example 4.2, DNA needs to be extracted from the sample to be tested. DNA extraction can be performed using a nucleic acid extraction or purification kit produced by Hangzhou Dilan Biotechnology Co., Ltd., catalog number DL-A-006.

[0094] 1. Preparation of PCR reaction tubes (reagent preparation area) Determine the required number of reaction tubes n (number of samples + negative control + positive control); remove sterile purified water, PCR buffer, and enzymes, and thaw them on ice or at room temperature. All kit components must be briefly centrifuged before use. Each reaction system is shown in Table 15.

[0095] Table 15 Reaction system for multiplex quantitative digital PCR

[0096] Calculate the amount of each reagent (except for the sample / control) according to the number of reaction tubes n, and add them to the centrifuge tubes. Mix thoroughly (it is recommended to use a pipette to slowly and repeatedly mix while avoiding liquid splashing or generating a large number of bubbles). After short-term centrifugation, dispense 23 μL into each PCR reaction tube.

[0097] 2. Sample addition (sample processing area or sample addition area) Add 7 μL of the DNA sample to be tested, or the negative control or positive control sample, to the prepared PCR reaction tubes. After sealing the tubes with a membrane, centrifuge briefly and transfer them to the corresponding positions on the Little Turtle fully automated microdroplet preparation instrument. Open the sealing membrane and add 2 mL of oil A and 1 mL of oil B to the corresponding positions on the oil tank plate, respectively. After placing the consumables (chip, pipette tip, waste container, etc.), start the instrument to prepare the droplets.

[0098] 3. PCR amplification Place the prepared droplet reaction chip in a PCR amplification instrument and perform the amplification reaction according to the conditions listed in Table 16 below, based on the edited sample information.

[0099] Table 16 Amplification reaction procedure for multiplex quantitative digital PCR

[0100] 4. Chip reading Transfer the amplified chip to a chip reader for reading. Select the FAM, VIC, ROX, CY5, and CY5.5 channels according to actual needs, choose the Direct Quantification (DQ) mode, and set the dilution factor to 4.286 (total volume of the detection system / sample volume in the reaction solution). After setting the sample name, start reading.

[0101] 5. Quality Control Standards The negative and positive controls of this kit must simultaneously meet the following conditions; otherwise, the experiment will be considered invalid and needs to be repeated: In a one-dimensional scatter plot, the positive control is clearly divided into upper and lower layers, with few or no discrete droplets in the middle (a "raining phenomenon"). The lower layer of the negative control is roughly the same as the positive control, and there are no discrete droplets compared to the upper part of the positive control.

[0102] 6. Data Analysis Open the read chip data using the BioAnalysiserSetup software. In the threshold line mode of the one-dimensional scatter plot, adjust the threshold lines for the positive and negative controls to be in the same position, ensuring that the positive and negative clusters in the positive control are clearly distinguishable, and that all points in the negative control are negative. Adjust the threshold line for the detection group to the same position as the positive and negative clusters, ensuring that the threshold line is located in a clearly defined blank area. If the threshold line can only be adjusted to a large number of discrete droplets, and the distinction between positive and negative droplets in the sample is not clear, it is recommended to troubleshoot the problem and repeat the experiment to ensure that the positive and negative results of the sample are clearly distinguishable. After adjusting the thresholds, the detection results of the chip will be displayed in the "Results List". The method for judging the results is shown in Table 17.

[0103] Table 17 Methods for Determining Results of Multiplex Quantitative Digital PCR

[0104] The droplet preparation instrument, PCR amplification instrument, and chip reader used in this invention are all products of Shanghai Xiaohaigui Technology Co., Ltd.'s BioDigital digital PCR system "Qing" series, which includes three instruments: 1) a fully automated sample processing system (Xiangzhu Medical Equipment Registration No. 20210021), model Loader Z200; 2) a PCR amplification instrument (Xiang Medical Equipment Registration No. 20212220277), model Cycler Z200; 3) a biochip reader (Xiang Medical Equipment Registration No. 20212220236), model Imager Z200.

[0105] Example 7 Performance indicators of multiplex fluorescent PCR and multiplex quantitative digital PCR kits 1. Performance indicators of the multiplex fluorescent PCR kit 1.1 Study on amplification efficiency Using the kit described in Example 4.1 of this invention, and following the method described in Example 5, the amplification efficiency was studied to verify the performance of the reagent. The detection substance was the plasmid prepared in Example 2. The results are shown in Table 18 below.

[0106] Table 18 Study on amplification efficiency

[0107] The amplification efficiency results showed that the amplification efficiency of each target gene and internal control was within the range of 90%-110%, which met the requirements.

[0108] 1.2 Limit of Detection Study (copies / mL) Using the kit described in Example 4.1 of this invention, and following the method in Example 5, the concentrations of four Aspergillus fungal plasmids (prepared in Example 2) were calibrated. The plasmid samples for each target gene were then diluted to 75 copies / mL. 1 mL of nucleic acid extraction reagent using magnetic beads was used for extraction. The results showed that Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus were detected at least 19 times in 20 replicates at 75 copies / mL (95% sample detection rate). The limit of detection for each target gene was 75 copies / mL. The results are shown in Table 19 below.

[0109] Table 19 Detection Limit (copies / mL) Experimental Results

[0110] 1.4 Model Equivalence Study Using the kit described in 4.1 of Example 4 of this invention, and following the method described in Example 5, the PCR was performed on the company's existing ABI 7500 fluorescence PCR instrument and Hongshi fluorescence PCR instrument. The results are shown in Table 20. The results of the ABI 7500 fluorescence PCR instrument and the Hongshi fluorescence PCR instrument are equivalent.

[0111] Table 20 Equivalent Models

[0112] 1.5 Real Sample Testing Ninety-one sputum samples were provided to our company by our research partner hospitals. DNA was extracted using an extraction kit from Hangzhou Dilan Biotechnology Co., Ltd. Nucleic acid was detected in the samples using the kit described in section 4.1 of Example 4 of this invention, following the method in Example 5. Of these, 45 samples were negative and 46 were positive. All negative samples were negative, indicating good specificity of the kit. Several positive samples showed mixed positive results for multiple bacterial species, indicating high sensitivity of the kit. The test results were consistent with the clinical laboratory tests performed on each sample, and the results were completely consistent with the actual situation. The results are shown in Tables 21 and 22 below.

[0113] Table 21 Detection Results of True Negative Samples

[0114] Table 22 Detection results of real positive samples

[0115] 1.6 Thermal Accelerated Stability The stability of the kit was studied using the kit described in Example 4.1 of this invention, following the method in Example 5. The stability of the kit was evaluated by accelerating the reaction at 37±0.5℃ for 0 days and 7 days. The results are shown in Table 23 below. The performance of the kit at 37±0.5℃ for 7 days meets the requirements.

[0116] Table 23 Results of accelerated assay at 37±0.5℃ using the reagent kit

[0117] Note: In the table, the specificity reference material refers to the specificity reference material prepared by the applicant company using the genomes of specific strains other than Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus, used to test the performance of the kit; P1~P3 refer to three parallel tests of the mixed positive plasmid of Aspergillus fumigatus and Aspergillus terreus, P4~P6 refer to three parallel tests of the mixed positive plasmid of Aspergillus flavus and Aspergillus terreus, and P7~P9 refer to three parallel tests of the mixed positive plasmid of Aspergillus niger and Aspergillus terreus; the detection limit reference material refers to the detection limit (slightly higher than the lowest detection limit) reference material prepared by the applicant company using the genomes of a mixed strain of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus, used to test the performance of the kit.

[0118] 2. Performance indicators of the multiplex quantitative digital PCR kit 2.1 Plasmid linearization To ensure sufficient reaction for multiplex quantitative digital PCR, the positive plasmid (prepared in Example 2) was linearized by restriction enzyme digestion. 10 ng / μL of plasmid was prepared and digested using the plasmid's unique restriction endonuclease site, EcoRI (EcoRI was purchased from New England Biolabs [NEB], catalog number: R0101V, 20000 units / mL). Preparation of the reaction system: EcoR Ⅰ: 1 μL; 10×Quick Cut Buffer: 5 μL; plasmid: 5 μL; add water to 50 μL.

[0119] Set the enzyme digestion program as follows: 37℃: 15min, 95℃: 5min, store at 4℃.

[0120] 2.2 Precision Study Using the kit described in Example 4.2 of this invention, the precision of the kit was studied according to the method in Example 6. Linearized plasmids at two different concentrations were detected, with 10 reproducible tests performed at each concentration: 1000 copies / μL and 100 copies / μL. The results are shown in Table 24 below. The mean and standard deviation of the detection results (concentration values) at each concentration were calculated, and then the CV value was calculated. The requirement was that the CV value be ≤10% at both the high and low concentrations. The detection results showed that all requirements were met.

[0121] Table 24 Results of 10 runs for each of the two concentrations of the precision reference material.

[0122] 2.3 Study on Linear Range Using the kit described in Example 4.2 of this invention (the difference being that the Aspergillus flavus probe is modified with a VIC fluorescent group, otherwise identical to Example 4.2), the linear range of the detection concentration of the kit was studied according to the method in Example 6. The standard linearized plasmid was serially diluted 4-fold, and a total of 7 concentrations (unit: copies / mL) were tested: 2.00E+07; 5.00E+06; 1.25E+06; 3.13E+05; 7.81E+04; 1.95E+04; 4.88E+03. Each concentration was tested three times, and the logarithmic correlation coefficient between the theoretical concentration and the average measured concentration was calculated. The results are shown in Table 25. The required R... 2 >0.980.

[0123] Table 25 Experimental Results of Linear Range

[0124] Correlation analysis showed that, as shown in Table 26, the measured values ​​of the four Aspergillus species were significantly positively correlated with the expected theoretical values; all R values... 2 >0.980, p<0.0001, the regression equations for each channel are shown in Table 26, indicating that the linear range of the reagent meets the validation criteria.

[0125] Table 26 Statistical table of regression analysis of the logarithmic values ​​of the theoretical concentration mean and the logarithmic value of the measured concentration mean for each channel.

[0126] 2.4 Detection Limit Test Using the kit described in Example 4.2 of this invention, the detection limit of the kit was studied according to the method in Example 6. Using the lowest detection limit reference standard provided by Dylan Biotechnology, five extractions were performed at the set lowest detection limit concentration and lower concentrations. The product concentrations were measured and recorded (copies / μL) (Table 27). At a plasmid concentration of 300 copies / mL (0.3 copies / μL), the positive detection rate of each target gene was 100%. At a lower plasmid concentration of 250 copies / mL (0.25 copies / μL), the positive detection rate of each target gene was 20%-80%. Only 100% detection was acceptable; therefore, the detection limit of this kit is 300 copies / mL.

[0127] Table 27 Reference Results for Minimum Detection Limit

[0128] 2.5 Real Sample Testing Using the kit described in section 4.2 of Example 4 of this invention, and following the method in Example 6, nucleic acid was detected in real clinical samples. Forty-six sputum-positive samples (identified by the above-mentioned multiplex fluorescent PCR method) provided by a research collaborating hospital were used. DNA was extracted using the nucleic acid extraction kit developed by Hangzhou Dilan Biotechnology Co., Ltd., and detected using the four Aspergillus multiplex quantitative digital PCR kit of this invention. The results are shown in Table 28 below. Regarding the detection results of real clinical samples, many samples showed mixed positive results for multiple targets, similar to the detection results of the multiplex fluorescent PCR kit of this invention. The two kits, using different methods, showed similar results, mutually confirming each other. The detection results meet the design requirements of the kit.

[0129] Table 28 Detection Results of Positive Samples

[0130] Example 8 Quality control product concentration optimization Negative control: The negative control is a plasmid containing a human housekeeping gene fragment, synthesized by a biotechnology company, dissolved and diluted for later use. The nucleotide sequence of the internal control DNA fragment is shown in SEQ ID NO.30.

[0131] Positive control samples (prepared from Example 2): plasmids containing Aspergillus fumigatus DNA fragments, plasmids containing Aspergillus flavus DNA fragments, plasmids containing Aspergillus niger DNA fragments, plasmids containing Aspergillus terreus DNA fragments, and plasmids containing internal reference DNA fragments were synthesized by a biotechnology company, dissolved, diluted, and used for later use. The nucleotide sequences of the Aspergillus fumigatus gene fragments in the above positive control plasmids are shown in SEQ ID NO.18, the Aspergillus flavus gene fragments in the above positive control plasmids are shown in SEQ ID NO.21, the Aspergillus niger gene fragments in the above positive control plasmids are shown in SEQ ID NO.24, the Aspergillus terreus gene fragments in the above positive control plasmids are shown in SEQ ID NO.27, and the internal reference DNA fragments in the above positive control plasmids are shown in SEQ ID NO.30.

[0132] Each target gene was diluted to 5 × 10⁻⁶. -6 The concentration of each target gene copy was measured at ng / μL, using the same method as in Example 6, employing the kit described in section 4.2 of Example 4 (the difference being that the fluorescent group modified with the internal control probe was CY5.5, while the rest was the same as section 4.2 of Example 4). The results are shown in Table 29. Based on the detection results, 10... 7 The mass concentration corresponding to copies / μL, used as the stock solution concentration for quality control, was 0.11 ng / μL for Aspergillus fumigatus, 0.16 ng / μL for Aspergillus flavus, 0.13 ng / μL for Aspergillus niger, 0.14 ng / μL for Aspergillus terreus, and 0.19 ng / μL for the internal control, respectively. Based on the detection results, the plasmid stock solution was diluted to the corresponding concentrations, serially diluted 20,000 times after equal-proportional mixing, and the concentrations of each target gene were measured, controlled within the range of 100 ± 20 copies / μL, as shown in Table 30.

[0133] Table 29 5×10 -6 Plasmid copy concentration (ng / μL) detection

[0134] Table 30 shows the detection of mixed plasmid concentrations (copies / μL).

[0135] Example 9 Study on the preservation matrix of quality control products A DNase inhibitor was added to the quality control sample, and a thermal accelerated test (37±0.5℃) was performed. The stability was compared with that at refrigerated temperatures (2-8℃). The specific method was the same as in Example 6, using the kit described in section 4.2 of Example 4 (the difference being that the fluorescent group modified by the internal control probe was CY5.5, while the rest was the same as section 4.2 of Example 4). The experimental results showed that the positive control sample of the multiplex quantitative digital PCR kit of the present invention had poor stability at 37±0.5℃ for 7 days, with a CV value between 83% and 98%. The stability at 2-8℃ was better than that at 37±0.5℃ (between 9% and 39%), but it still did not meet the design requirements. After adding the DNase inhibitor, the degradation of the quality control sample decreased, and the overall CV decreased. At 37±0.5℃ for 7 days, the CV decreased to between 53% and 63%, and at 2-8℃ for 7 days, it was between 8% and 35%. Although the quality control material could not withstand environments of 37±0.5℃ and 2-8℃, the addition of a DNase inhibitor reduced the degradation of the quality control material, indicating the negative impact of DNase on the quality control material in these two environments. Therefore, the necessity of using a DNase inhibitor is demonstrated (results are shown in Table 31).

[0136] Table 31. Preservation effect of DNase inhibitors (copies / μL)

[0137] Example 10 Study on the amount of DNA enzyme inhibitor added In Example 9, the addition of a DNase inhibitor improved the preservation of the quality control sample. Therefore, further experiments were conducted to study the dosage used, specifically the volume ratio of the DNase inhibitor. The specific method was the same as in Example 6, using the kit described in section 4.2 of Example 4 (the difference being that the fluorescent group modified by the internal control probe was CY5.5, while the rest was the same as section 4.2 of Example 4). The results showed that adding 2 / 1000 to 3 / 1000 (v / v) volume of DNase inhibitor resulted in a smaller overall CV value compared to 1 / 1000, and the detection concentration was relatively high after 7 days of preservation, indicating a better preservation effect. Among these, 3 / 1000 (v / v) volume of DNase inhibitor showed the best effect, as detailed in Table 32.

[0138] Table 32 Results of thermally accelerated detection of DNase inhibitors at different volume ratios (copies / μL)

[0139] Example 11 Stability Study of Quality Control Products The plasmid dilution buffer used for quality control samples consisted of salmon sperm DNA (Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number 31149-10G-F, white powder), a DNase inhibitor (Beijing Biolabs Technology Co., Ltd., catalog number: HR9112), and 0.1×TE. The final concentration of salmon sperm DNA in the plasmid dilution buffer was 500 ng / μL, and the volume ratio of DNase inhibitor to plasmid dilution buffer was 3:1000. After dissolving the dry powder of each quality control sample, the concentration was corrected to 100±20 copies / μL, and then the above plasmid dilution buffer was added. The freeze-thaw stability and long-term real-time stability of the quality control plasmid were tested using the same method as in Example 6, employing the kit described in section 4.2 of Example 4. The results showed that when the quality control plasmid was stored at -20℃±10℃ for ≤4 freeze-thaw cycles, with a concentration of 100±20 copies / μL, the CV value could be controlled within 3%-10%, meeting the requirement of CV≤15%. When the quality control plasmid was stored at -20℃±10℃ for 60 days, with a concentration of 100±20 copies / μL, the CV value could be controlled within 3%-12%, meeting the requirement of CV≤15%. Specific results are shown in Tables 33 and 34.

[0140] Table 33 Freeze-thaw stability of positive control samples

[0141] Table 34 Long-term real-time stability of positive control samples at -20℃±10℃

[0142] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer-probe combination capable of simultaneously qualitatively or quantitatively detecting four Aspergillus species, characterized in that, Includes the following primer-probe combinations: The primer-probe combination for detecting Aspergillus fumigatus includes primer pairs with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2 and probes with nucleotide sequences as shown in SEQ ID NO.3; The primer-probe combination for detecting Aspergillus flavus includes primer pairs with nucleotide sequences as shown in SEQ ID NO.4 and SEQ ID NO.5 and probes with nucleotide sequences as shown in SEQ ID NO.6; The primer-probe combination for detecting Aspergillus niger includes primer pairs with nucleotide sequences as shown in SEQ ID NO.7 and SEQ ID NO.8 and probes with nucleotide sequences as shown in SEQ ID NO.9; The primer-probe combination for detecting Aspergillus terreus includes primer pairs with nucleotide sequences as shown in SEQ ID NO.10 and SEQ ID NO.11 and probes with nucleotide sequences as shown in SEQ ID NO.

12.

2. The primer-probe combination according to claim 1, characterized in that, The probes for detecting Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus are modified with fluorescent and quenching groups at both ends, respectively. The fluorescent group includes any one of FAM, ROX, VIC, HEX, CY5, CY5.5, or Quasar705; the quenching group includes any one of BHQ1, BHQ2, BHQ3, or MGB. When detecting multiple Aspergillus species simultaneously, the fluorescent groups modified at both ends of the probes for different Aspergillus species are different.

3. The application of the primer-probe combination of claim 1 in the preparation of multiplex fluorescent PCR detection and / or multiplex quantitative digital PCR detection products.

4. A multiplex fluorescent PCR detection kit capable of simultaneously qualitatively detecting four Aspergillus species, characterized in that, It comprises the following components: a detection mixture containing the primer-probe combination of claim 1, an amplification reaction solution, a negative control, and a positive control.

5. The multiplex fluorescent PCR detection kit according to claim 4, characterized in that, The detection mixture includes detection mixture 1 and detection mixture 2. Detection mixture 1 includes the primer-probe combination for detecting Aspergillus fumigatus, the primer-probe combination for detecting Aspergillus flavus, the primer-probe combination for detecting Aspergillus niger, and the primer-probe combination for detecting the internal reference GAPDH. Detection mixture 2 includes the primer-probe combination for detecting Aspergillus terreus and the primer-probe combination for detecting the internal reference. The primer-probe combination for detecting the internal reference includes primer pairs with nucleotide sequences as shown in SEQ ID NO.13 and SEQ ID NO.14 and probes with nucleotide sequences as shown in SEQ ID NO.

15. The final concentration of each primer in detection mixture 1 is 0.2 μM, and the final concentration of each probe is 0.15 μM. The final concentration of each primer in detection mixture 2 is 0.2 μM, and the final concentration of each probe is 0.15 μM.

6. A multiplex quantitative digital PCR detection kit capable of simultaneously quantifying four Aspergillus species, characterized in that, It comprises the following components: a detection mixture containing the primer-probe combination of claim 1, an enzyme, a buffer, a negative control, a positive control, and nuclease-free water.

7. The multiplex fluorescent PCR detection kit according to claim 4 or the multiplex quantitative digital PCR detection kit according to claim 6, characterized in that, The negative control is a recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO. 30; the positive control is a mixture containing Aspergillus fumigatus recombinant plasmid, Aspergillus flavus recombinant plasmid, Aspergillus niger recombinant plasmid, Aspergillus terreus recombinant plasmid, and an internal reference recombinant plasmid; the Aspergillus fumigatus recombinant plasmid contains the Aspergillus fumigatus fragment with the nucleotide sequence shown in SEQ ID NO. 18, the Aspergillus flavus recombinant plasmid contains the Aspergillus flavus fragment with the nucleotide sequence shown in SEQ ID NO. 21, the Aspergillus niger recombinant plasmid contains the Aspergillus niger fragment with the nucleotide sequence shown in SEQ ID NO. 24, the Aspergillus terreus recombinant plasmid contains the Aspergillus terreus fragment with the nucleotide sequence shown in SEQ ID NO. 27, and the internal reference recombinant plasmid contains the internal reference fragment with the nucleotide sequence shown in SEQ ID NO.

30.

8. The multiplex fluorescent PCR detection kit according to claim 4 or the multiplex quantitative digital PCR detection kit according to claim 6, characterized in that, Negative and positive controls contain DNA protectants, including salmon sperm DNA and DNA nuclease inhibitors.

9. The multiplex fluorescent PCR detection kit according to claim 4, characterized in that, The amplification reaction program was 37℃ for 5 min; 95℃ for 5 min; 95℃ for 15 s and 60℃ for 30 s, for 45 cycles; The result determination method is as follows: Negative and positive controls should simultaneously meet the following conditions; otherwise, the test is considered invalid and needs to be repeated: If the negative control Ct value is >40 or "Undetermined", and the internal reference Ct value is <44, and the positive control Ct value is ≤40, and the internal reference Ct value is <44; If the test is valid, the test sample is then determined: If the sample Ct value is ≤40, the internal reference Ct value is <44, or no internal reference Ct value is detected, it indicates a positive result; if the sample Ct value is >40 or "Undetermined", and the internal reference Ct value is <44, it indicates a negative result.

10. The multiplex quantitative digital PCR detection kit according to claim 6, characterized in that, The final concentration of each primer in the detection mixture is 0.2 μM, and the final concentration of each probe is 0.15 μM. The amplification reaction program was as follows: oil curing at 50℃ for 5 min; pre-denaturation at 95℃ for 5 min; cyclic reaction at 95℃ for 15 s and 60℃ for 30 s, for 45 cycles; and holding at 25℃. The result determination method is as follows: Negative and positive controls should simultaneously meet the following conditions; otherwise, the test is considered invalid and needs to be repeated: The one-dimensional scatter plot shows that the positive control is clearly divided into upper and lower layers, and the negative control signal points are concentrated in the lower layer of the positive control, where the fluorescence intensity is consistent. There are no discrete droplets compared to the upper part of the positive control. If the test is valid, the sample to be tested is determined as follows: If the number of positive internal reference points is <3, the quantitative result is invalid; if the number of positive sample points is <1 and the number of positive internal reference points is ≥3, the result is negative; if the number of positive sample points is ≥1 and the number of positive internal reference points is ≥3, the result is positive, and the quantitative value is output.

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