Primer probe set and kit for multiple fluorescent PCR (Polymerase Chain Reaction) and / or multiple quantitative digital PCR detection of rhizopus, mucor, rhizomucor, Aureobasidium and Cunninghaga sp.

Abstract

The invention belongs to the technical field of in-vitro molecular detection of pathogenic microorganisms, and particularly relates to a primer probe group and a kit for multiple fluorescent PCR (Polymerase Chain Reaction) and / or multiple quantitative digital PCR detection of rhizopus, mucor, rhizomucor, trachycarpus and cunningham fungi. The primer probe group disclosed by the invention can be used for realizing'single-tube quintuplet 'qualitative detection by adopting a multiple fluorescent PCR method, can also be used for realizing'five-target synchronous absolute quantification' by adopting a multiple quantitative digital PCR method, and can also be used for combining the'single-tube quintuplet 'qualitative detection of the multiple fluorescent PCR method with the'five-target synchronous absolute quantification' of the multiple quantitative digital PCR method. The primer probe group and the kit provided by the invention have the characteristics of high specificity, high sensitivity, short detection period, high tolerance to a complex sample matrix and the like, and an integrated molecular detection solution is provided for early and accurate diagnosis of clinical invasive hair mold.

Description

Technical Field

[0001] This invention belongs to the field of in vitro molecular detection technology for pathogenic microorganisms, specifically relating to primer and probe sets and kits for multiplex fluorescent PCR and / or multiplex quantitative digital PCR detection of fungi in the genera Rhizopus, Mucor, Rhizopus, Rhizopus, and Cladosporium. Background Technology

[0002] Mucormycosis is an invasive fungal infection caused by fungi in the order Mucorales, primarily affecting immunocompromised patients and causing both localized and disseminated infections. Among Mucorales, *Rhizopus*, *Mucor*, *Rhizomucor*, *Lichtheimia*, and *Cunninghamella* are the main pathogens causing invasive mucormycosis. Metabolic disorders such as poorly controlled diabetes, diabetic ketoacidosis, and metabolic acidosis; compromised immune function such as hematologic malignancies, bone marrow or stem cell transplantation, organ transplantation, AIDS, severe malnutrition, use of glucocorticoids or immunosuppressants; and impaired skin barrier function such as burns and injuries all increase the risk of infection by Mucorales fungi. Early, rapid, and accurate etiological detection is crucial for guiding precise antifungal therapy (such as amphotericin B and its lipid preparations, which are the main treatment drugs, although different genera may have different sensitivities to these drugs) and improving patient prognosis. Delayed diagnosis and treatment significantly increase the risk of death.

[0003] Early clinical and imaging manifestations of mucormycosis lack specificity, making clinical diagnosis difficult. Therefore, achieving rapid early diagnosis of mucormycosis is crucial. However, traditional morphological identification of Mucorales fungi is highly demanding, as some species share highly similar morphological characteristics and are common environmental contaminants. Direct microscopic examination of specimens and histopathological sections are necessary for identification, but microscopic examination has low sensitivity, and histopathological sections often fail to distinguish Mucor hyphae. Furthermore, in the field of molecular biology, reported patented multiplex PCR methods for detecting mucormycosis mostly focus on detecting the 2-4 most common genera (Rhizopus, Mucor, Rhizomucor, Lichtheimia), rarely including the Cunninghamella genus, or failing to comprehensively cover the most clinically important and morphologically difficult-to-distinguish pathogens of these five genera. For example, patent CN116445647A, "Reagent Kit for Detecting Aspergillus, Rhizopus, and Mucor," only includes Rhizopus and Mucor genera; patent CN 112980997 B, "Primers and Probes for Invasive Mucor Pathogens and Their Implementation Methods and Detection Systems," does not include *Aureobasidium spp.* Currently, there is a lack of a molecular detection method that can simultaneously cover all five genera of clinically important mucor pathogens, is rapid and efficient, highly sensitive, specific, has high throughput, and is suitable for trace sample detection. Traditional methods are slow and inaccurate; existing singlet or low-multiplex multiplex PCR methods are inefficient, have incomplete coverage, or limited sensitivity; and the potential of dPCR in the field of multiplex fungal detection has not yet been fully explored to address this specific clinical need.

[0004] Currently, there is a lack of rapid and efficient multiplex fluorescent PCR and multiplex quantitative digital PCR methods for the combined detection of nucleic acids from fungi of the genera Rhizopus, Mucor, Rhizopus, Rhizopus, and Cladosporium. Summary of the Invention

[0005] The purpose of this invention is to provide primer and probe sets and kits for the detection of fungi in the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Pheromone*, and *Clostridium* using multiplex fluorescent PCR and / or multiplex quantitative digital PCR. The primer and probe sets described in this invention enable sensitive and specific combined qualitative and / or quantitative detection of fungi in the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Pheromone*, and *Clostridium*, exhibiting high specificity, high sensitivity, speed, and efficiency.

[0006] This invention provides primer and probe sets for the detection of fungi in the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Pterygospermum*, and *Clostridium* using multiplex fluorescent PCR and / or multiplex quantitative digital PCR. These include primers dl-RHI-F1 and dl-RHI-R1 and probe dl-RHI-P1 for detecting *Rhizopus* fungi, primers dl-MUC-F1 and dl-MUC-R1 and probe dl-MUC-P1 for detecting *Mucor* fungi, and primers dl-MUC-F1 and dl-MUC-R1 and probe dl-MUC-P1 for detecting *Rhizopus* fungi. Primers dl-RHIZO-F1, dl-RHIZO-R1 and probe dl-RHIZO-P1 for detecting fungi, primers dl-ABS-F1, dl-ABS-R1 and probe dl-ABS-P1 for detecting fungi of the genus *Strombyx*, and primers dl-CUN-F1, dl-CUN-R1 and probe dl-CUN-P1 for detecting fungi of the genus *Strombyx*; the nucleotide sequence of dl-RHI-F1 is shown in SEQ. The nucleotide sequence of dl-RHI-R1 is shown in SEQ ID NO.1; the nucleotide sequence of dl-RHI-P1 is shown in SEQ ID NO.3; the nucleotide sequence of dl-MUC-F1 is shown in SEQ ID NO.4; the nucleotide sequence of dl-MUC-R1 is shown in SEQ ID NO.5; the nucleotide sequence of dl-MUC-P1 is shown in SEQ ID NO.6; the nucleotide sequence of dl-RHIZO-F1 is shown in SEQ ID NO.7; the nucleotide sequence of dl-RHIZO-R1 is shown in SEQ ID NO.8; the nucleotide sequence of dl-RHIZO-P1 is shown in SEQ ID NO.9; the nucleotide sequence of dl-ABS-F1 is shown in SEQ ID NO.10; the nucleotide sequence of dl-ABS-R1 is shown in SEQ ID NO.11; the nucleotide sequence of dl-ABS-P1 is shown in SEQ ID NO.12; the nucleotide sequence of dl-CUN-F ... As shown in ID NO.13; the nucleotide sequence of dl-CUN-R1 is shown in SEQ ID NO.14; the nucleotide sequence of dl-CUN-P1 is shown in SEQ ID NO.15; the 5' ends of dl-RHI-P1, dl-MUC-P1, dl-RHIZO-P1, dl-ABS-P1 and dl-CUN-P1 are labeled with fluorescent groups, and the 3' ends are labeled with quenching groups.

[0007] Preferably, the primer-probe set further includes internal reference primers dl-GH-F1 and dl-GH-R1 and probe dl-GH-P1; the nucleotide sequence of dl-GH-F1 is shown in SEQ ID NO.16; the nucleotide sequence of dl-GH-R1 is shown in SEQ ID NO.17; the nucleotide sequence of dl-GH-P1 is shown in SEQ ID NO.18; the 5' end of dl-GH-P1 is labeled with a fluorescent group, and the 3' end is labeled with a quencher group.

[0008] Preferably, the fluorescent group is selected from any one of FAM, ROX, Texas-Red, HEX, VIC, JOE, TET, CY5, Quasar705, CY5.5, ATTO425, and AF405; and the quenching group is selected from any one of BHQ1, BHQ2, BHQ3, and MGB.

[0009] The present invention also provides the application of the primer and probe sets described in the above technical solutions in the preparation of kits for multiplex fluorescent PCR and / or multiplex quantitative digital PCR to detect fungi of the genera Rhizopus, Mucor, Rhizopus, Rhizopus, and Clematis spp., or kits for detecting infectious diseases caused by fungi of the genera Rhizopus, Mucor, Rhizopus, Rhizopus, and Clematis spp.

[0010] This invention also provides a kit for detecting multiplex fluorescent PCR and / or multiplex quantitative digital PCR of fungi in the genera Rhizopus, Mucor, Rhizopus, Rhizopus, and Clematis, including the primer and probe set and reaction solution described in the above technical solution.

[0011] Preferably, the reaction solution includes multiplex fluorescent PCR reaction solution and / or multiplex quantitative digital PCR reaction solution.

[0012] Preferably, the kit further includes a multiplex fluorescent PCR positive control and a multiplex fluorescent PCR negative control, and / or the kit further includes a multiplex quantitative digital PCR reaction solution positive control.

[0013] Preferably, the multiplex fluorescent PCR positive control includes a cloning plasmid and an internal reference plasmid containing the nucleotide sequences of fungal genes from the gene gene *Rhizopus*, *Mucor*, *Rhizopus*, *Pterygospermum*, and *Clostridium clematis*, respectively; the multiplex fluorescent PCR negative control includes an internal reference plasmid; and the multiplex quantitative digital PCR reaction solution positive control includes a cloning plasmid containing the nucleotide sequences of fungal genes from the gene ...

[0014] Preferably, the cloning plasmids containing gene nucleotide sequences of Rhizopus, Mucor, Rhizopus, Rhizopus, and Cladosporium hainanense fungi respectively include cloning plasmids containing Rhizopus gene nucleotide sequences as shown in SEQ ID NO.21, Rhizopus gene nucleotide sequences as shown in SEQ ID NO.24, Rhizopus gene nucleotide sequences as shown in SEQ ID NO.27, Rhizopus gene nucleotide sequences as shown in SEQ ID NO.30, and Cladosporium hainanense gene nucleotide sequences as shown in SEQ ID NO.33; the internal reference plasmid contains the human housekeeper gene nucleotide sequence as shown in SEQ ID NO.36.

[0015] Preferably, when using multiplex fluorescent PCR to detect fungi of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Phyllostachys*, and *Clostridium*, the primer concentration is 0.2 μM and the probe concentration is 0.15 μM; when using multiplex quantitative digital PCR to detect fungi of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Phyllostachys*, and *Clostridium*, the primer concentration is 0.3 μM and the probe concentration is 0.15 μM.

[0016] This invention provides primer and probe sets for multiplex fluorescent PCR and / or multiplex quantitative digital PCR detection of *Rhizopus*, *Mucor*, *Rhizopus*, *Phyllostachys*, and *Clostridium*. The primer and probe sets of this invention contain specific primers and corresponding probe sets targeting the five common pathogenic fungi of the *Mucor* family. Based on this primer and probe set, this invention establishes two compatible detection pathways: qualitative detection using multiplex fluorescent PCR (single-tube or double-tube five-target assay) or simultaneous absolute quantification of five targets on a single chip using multiplex quantitative digital PCR; alternatively, qualitative detection using multiplex fluorescent PCR (single-tube or double-tube five-target assay) can be combined with simultaneous absolute quantification of five targets on a single chip using multiplex quantitative digital PCR. Experimental results show that, using the primer and probe set of this invention, the detection limit for the five *Mucor* targets (covering 14 *Mucor* species) using multiplex fluorescent PCR is 100 copies / mL; the detection limit for the nucleic acids of the five *Mucor* targets using multiplex quantitative digital PCR is 0.3 copies / μL. Based on the primer and probe set of this invention, a kit is developed that provides two methodologies for the specific and sensitive detection of nucleic acids in Rhizopus, Mucor, Rhizopus, Rhizopus, and Clematis spp. Fungi. This kit is suitable for early typing and qualitative detection as well as precise quantitative detection of suspected Rhizopus, Mucor, Rhizopus, Rhizopus, and Clematis spp. infections in clinical practice, providing an integrated molecular detection solution for the early and accurate diagnosis of invasive mucormycosis.

[0017] Specifically, the primer-probe set of the present invention has the following beneficial effects: (1) It can specifically detect nucleic acid molecules of five Mucor genera, namely Mucor, Rhizopus, Rhizopus and Rhizopus, in one instrument or on one chip using multiple pairs of primers and probes (fluorescent PCR) and / or quantitative detection. (2) High specificity: No cross-reactivity with common clinical fungi (such as Aspergillus fumigatus, Candida albicans, Candida tropicalis, etc.) and bacteria; (3) High sensitivity: Multiplex fluorescent PCR: It can still be detected when the content of the target gene of any of the five Mucor genera is 100 copies / mL; Multiplex quantitative digital PCR: It can detect down to a single copy (only 1 positive spot). (4) Short detection cycle: 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) High tolerance to complex sample matrices: Multiplex fluorescent PCR and / or quantitative digital PCR are not sensitive to PCR inhibitors; (6) Multiplex quantitative digital PCR can quantify the target without the need to establish a standard curve, and the method is simple; (7) The results of multiplex quantitative digital PCR are clear and easy to interpret, and the positive and negative results are clearly distinguished. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The amplification curve results of primer specificity test (multiplex fluorescent PCR) provided for this invention are shown in the following figures: The upper figure shows the results of *Rhizopus*, *Mucor*, and *Rhizopus* and the internal reference; the lower figure shows the results of *Rhizopus*, *Clostridium*, and the internal reference. Figure 2 The results of the test (fluorescent PCR method) for two concentrations of *Rhizopus*, *Mucor*, *Rhizopus*, *Rhizopus* and *Hymenopterus* provided by this invention, with detection limits of 1000 copies / mL and 100 copies / mL; Figure 3 The quantitative digital PCR precision test results provided for this invention are as follows: a one-dimensional scatter plot of 10 replicates in each of the 5 channels, with a precision concentration of 100 copies / μL as an example. Figure 4 The accuracy test results provided for this invention are as follows: taking a concentration of 10 copies / μL as an example, 10 replicates of 1D scatter plots for each of the 5 channels; Figure 5 The graph shows the test results for the detection limit of quantitative digital PCR provided by this invention. Detailed Implementation

[0020] This invention provides primer and probe sets for the detection of fungi in the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Pterygospermum*, and *Clostridium* using multiplex fluorescent PCR and / or multiplex quantitative digital PCR. These include primers dl-RHI-F1 and dl-RHI-R1 and probe dl-RHI-P1 for detecting *Rhizopus* fungi, primers dl-MUC-F1 and dl-MUC-R1 and probe dl-MUC-P1 for detecting *Mucor* fungi, and primers dl-MUC-F1 and dl-MUC-R1 and probe dl-MUC-P1 for detecting *Rhizopus* fungi. Primers dl-RHIZO-F1, dl-RHIZO-R1 and probe dl-RHIZO-P1 for detecting fungi, primers dl-ABS-F1, dl-ABS-R1 and probe dl-ABS-P1 for detecting fungi of the genus *Strombyx*, and primers dl-CUN-F1, dl-CUN-R1 and probe dl-CUN-P1 for detecting fungi of the genus *Strombyx*; the nucleotide sequence of dl-RHI-F1 is shown in SEQ. The nucleotide sequence of dl-RHI-R1 is shown in SEQ ID NO.1; the nucleotide sequence of dl-RHI-P1 is shown in SEQ ID NO.3; the nucleotide sequence of dl-MUC-F1 is shown in SEQ ID NO.4; the nucleotide sequence of dl-MUC-R1 is shown in SEQ ID NO.5; the nucleotide sequence of dl-MUC-P1 is shown in SEQ ID NO.6; the nucleotide sequence of dl-RHIZO-F1 is shown in SEQ ID NO.7; the nucleotide sequence of dl-RHIZO-R1 is shown in SEQ ID NO.8; the nucleotide sequence of dl-RHIZO-P1 is shown in SEQ ID NO.9; the nucleotide sequence of dl-ABS-F1 is shown in SEQ ID NO.10; the nucleotide sequence of dl-ABS-R1 is shown in SEQ ID NO.11; the nucleotide sequence of dl-ABS-P1 is shown in SEQ ID NO.12; the nucleotide sequence of dl-CUN-F ... As shown in ID NO.13; the nucleotide sequence of dl-CUN-R1 is shown in SEQ ID NO.14; the nucleotide sequence of dl-CUN-P1 is shown in SEQ ID NO.15; the 5' ends of dl-RHI-P1, dl-MUC-P1, dl-RHIZO-P1, dl-ABS-P1 and dl-CUN-P1 are labeled with fluorescent groups, and the 3' ends are labeled with quenching groups.In a specific embodiment, the primer-probe set further includes internal reference primers dl-GH-F1 and dl-GH-R1 and probe dl-GH-P1; the nucleotide sequence of dl-GH-F1 is shown in SEQ ID NO.16; the nucleotide sequence of dl-GH-R1 is shown in SEQ ID NO.17; the nucleotide sequence of dl-GH-P1 is shown in SEQ ID NO.18; the 5' end of dl-GH-P1 is labeled with a fluorescent group, and the 3' end is labeled with a quencher group. In a specific embodiment, the fluorescent group includes any one selected from FAM, ROX, Texas-Red, HEX, VIC, JOE, TET, CY5, Quasar705, CY5.5, ATTO425, and AF405; the quencher group is selected from any one of BHQ1, BHQ2, BHQ3, and MGB. This invention designs multiple pairs of specific primers based on ribosomal DNA fragments from fungi of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Phyllostachys*, and *Clostridium*. When the primer-probe set of this invention is used for multiplex fluorescent PCR detection, this invention determines whether specific PCR products are formed by detecting the intensity of the fluorescence signal and the shape of the amplification curve. Based on the difference in melting temperature of different sequences, it rapidly identifies fungi of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Phyllostachys*, and *Clostridium*, thereby determining whether the tested sample contains the target DNA fragment and what type of target DNA fragment it is. The primer-probe set of this invention can be used for qualitative, rapid, and efficient detection of nucleic acid molecules from the five genera *Rhizopus*, *Mucor*, *Rhizopus*, *Phyllostachys*, and *Clostridium*, distinguishing the presence of each genera in clinical samples. Digital PCR (dPCR), as a third-generation PCR technology, has extremely high sensitivity, detecting even single copies of nucleic acid, and has significant advantages in detecting early pathogen infections. Depending on the formation method of the reaction unit, dPCR is divided into chip digital PCR (cdPCR) and droplet digital PCR (ddPCR). The primer and probe set of this invention can be used for multiplex quantitative digital PCR detection of fungi in the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Phyllostachys*, and *Clostridium*. In a specific embodiment, chip digital PCR is used. This method distributes a single tube of amplification reaction system into tens of thousands of microwells (or droplets), forming a multi-well (droplet) amplification reaction system. The copy number of the target nucleic acid molecule is calculated based on the ratio of droplets in the positive and negative wells after the reaction. This invention achieves random dispersion of nucleic acid samples in the PCR reaction, allowing for sufficient separation between the microwells containing positive and negative droplets, reducing background signals, enhancing the amplification signal of positive targets, and greatly improving the detection sensitivity of low-copy samples.The primer and probe set of this invention can be used for the rapid and efficient quantitative detection of nucleic acid molecules from five genera of Mucor: *Rhizopus*, *Mucor*, *Rhizopus*, *Rhizopus*, and *Clostridium*. Experimental results show that the primer and probe set of this invention has high specificity and does not cross-react with common human and clinical bacteria and fungi. It can simultaneously and efficiently amplify the five Mucor genera using specific primers and probes in both fluorescent PCR and quantitative digital PCR methods without interference or cross-reaction, overcoming the challenge of using universal multi-target primer and probe systems across different methodologies.

[0021] This invention also provides the application of the primer and probe set described above in the preparation of kits for multiplex fluorescent PCR and / or multiplex quantitative digital PCR to detect fungi of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Pheromone*, and *Clostridium*, or kits for detecting infectious diseases caused by fungi of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Pheromone*, and *Clostridium*. The primer and probe set of this invention enables rapid, accurate, and simultaneous detection of fungi from five genera of the order *Mucorales*, and can simultaneously achieve qualitative and quantitative detection of pathogens from the five *Mucor* genera, exhibiting high specificity and sensitivity.

[0022] This invention also provides a kit for detecting multiplex fluorescent PCR and / or multiplex quantitative digital PCR of fungi in the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Pheromone*, and *Clostridium*, comprising the primer and probe sets and reaction solutions described in the above-mentioned technical solutions. The kit of this invention enables multiplex qualitative and / or quantitative detection of fungi in the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Pheromone*, and *Clostridium*, and can also be used for the diagnosis of diseases caused by fungi in these genera. In a specific embodiment, the reaction solution includes multiplex fluorescent PCR reaction solution and / or multiplex quantitative digital PCR reaction solution. In a specific embodiment, the multiplex fluorescent PCR reaction solution can be Hieff Unicon® Universal TaqMan multiplex qPCR master mix (UDG plus), catalog number 11212ES08, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., which can improve the sensitivity of amplification. In a specific embodiment, the multiplex quantitative digital PCR reaction solution can be Multiplex qPCR mix, catalog number DL-D-001, purchased from Hangzhou Dilan Biotechnology Co., Ltd. This reaction solution has good reaction performance; under amplification with this solution, positive and negative results will be normally separated, and the signal-to-noise ratio is suitable. In a specific embodiment, the kit also includes a multiplex fluorescent PCR positive control and a multiplex fluorescent PCR negative control, and / or the kit also includes a multiplex quantitative digital PCR reaction solution positive control. In a specific embodiment, the multiplex fluorescent PCR positive control includes cloning plasmids and internal reference plasmids containing nucleotide sequences of fungal genes from the gene gene *Rhizopus*, *Mucor*, *Rhizopus*, *Phyllostachys*, and *Clostridium*, respectively; the multiplex fluorescent PCR negative control includes an internal reference plasmid; the multiplex quantitative digital PCR reaction solution positive control includes cloning plasmids containing nucleotide sequences of fungal genes from the gene ... In a specific embodiment, the cloning plasmids containing gene nucleotide sequences of Rhizopus, Mucor, Rhizopus, Rhizopus, and Cladosporium hainanense fungi respectively include cloning plasmids containing Rhizopus gene nucleotide sequences as shown in SEQ ID NO. 21, Rhizopus gene nucleotide sequences as shown in SEQ ID NO. 24, Rhizopus gene nucleotide sequences as shown in SEQ ID NO. 27, Rhizopus gene nucleotide sequences as shown in SEQ ID NO. 30, and Cladosporium hainanense gene nucleotide sequences as shown in SEQ ID NO. 33; the internal reference plasmid contains the human housekeeper gene nucleotide sequence as shown in SEQ ID NO. 36.In a specific embodiment, the primer-probe set can be added to the reaction system in the form of a mixture. The five primer-probe sets of this invention do not interfere with each other and can be detected in the same reaction tube or chip. In this embodiment, the multiplex fluorescent PCR detection device has four channels, so simultaneous detection is not performed in one reaction tube. However, the same reaction program can be used in two tubes simultaneously to detect the presence of the five *Mucor* fungi in the sample. In a specific embodiment, the sample to be tested includes bronchoalveolar lavage fluid. In specific embodiments, when using multiplex fluorescent PCR to detect fungi of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Phyllostachys*, and *Clostridium*, after experimental testing and repeated verification, the primer concentration was finally determined to be 0.2 μM and the probe concentration to be 0.15 μM. Similarly, when using multiplex quantitative digital PCR to detect fungi of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Phyllostachys*, and *Clostridium*, after experimental testing and repeated verification, the primer concentration was finally determined to be 0.3 μM and the probe concentration to be 0.15 μM.

[0023] Based on the primer and probe set or kit of this invention, this invention enables multiplex fluorescent PCR and / or multiplex quantitative digital PCR detection of five genera of Mucorales fungi. In other words, this invention also provides a method for multiplex fluorescent PCR and / or multiplex quantitative digital PCR detection of Rhizopus, Mucor, Rhizopus, Rhizopus, and Cladosporium. In a specific embodiment, the reaction system for the multiplex fluorescent PCR can be: 14 μL of nucleic acid amplification reaction solution, 5 μL of primer and probe detection mixture, and 6 μL of DNA sample or control, for a total volume of 25 μL. The final reaction procedure, determined through experimental testing, is shown in Table 16. In a specific embodiment, after experimental testing and verification, the reaction system for the multiplex quantitative digital PCR detection can be: 10 μL of digital PCR buffer, 0.5 μL of enzyme, 3 μL of primer and probe detection mixture, 7 μL of DNA sample or control, and 9.5 μL of nuclease-free water, for a total volume of 30 μL. The reaction procedure is shown in Table 19. The PCR reaction system and reaction procedure conditions defined in this invention, combined with the use of primer and probe sets, enable efficient multiplex fluorescent PCR and / or multiplex quantitative digital PCR detection.

[0024] 1. Nucleic acid testing Nucleic acid detection refers to detection methods and reagents that target deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in samples. Nucleic acid detection methods are diverse, including techniques such as fluorescence polymerase chain reaction (PCR) using nucleic acid amplification, DNA microarrays using nucleic acid hybridization, and fluorescence in situ hybridization (FISH).

[0025] 2. Fluorescent polymerase chain reaction (fluorescent PCR) method Fluorescent PCR is a PCR method that uses the change in fluorescence energy released by fluorescent labels during PCR primer amplification to directly reflect the change in the amount of PCR amplification products. The fluorescence signal variable is directly proportional to the amplification product variable. By collecting and analyzing the fluorescence light intensity, the original template can be qualitatively or quantitatively analyzed.

[0026] 3. Threshold cycle number (Ct value) In fluorescent PCR amplification, the fluorescence signal in the reaction tube reaches the point of exponential amplification through a cycle number of cycles. The main calculation method uses a threshold of 10 times the standard deviation of the fluorescence value during the first 3 to 15 cycles of amplification. The number of cycles when the fluorescence value exceeds this threshold is called the threshold cycle number, or Ct value. The Ct value is linearly negatively correlated with the initial logarithmic amount of the target DNA fragment.

[0027] 4. Quantitative digital PCR Quantitative digital PCR (qPCR) is a method that dilutes a standard PCR system containing nucleic acid templates, disperses the reaction into micro-units, and ensures that each reaction contains only one target nucleic acid molecule. This achieves single-molecule nucleic acid template PCR amplification. Utilizing the Poisson distribution principle, the copy number of the target nucleic acid molecule is calculated based on the ratio of negative to positive droplets after the reaction, thus achieving absolute quantification of single-molecule nucleic acid.

[0028] To further illustrate the present invention, the primer and probe sets and kits for multiplex fluorescent PCR and / or multiplex quantitative digital PCR detection of Rhizopus, Mucor, Rhizopus, Rhizopus, and Clavicipitaceae provided by the present invention are described in detail below with reference to embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1 Primer and probe screening (can be used for fluorescent PCR and quantitative digital PCR) 1. Primer and probe design and specificity testing within the Mucorales order. Primer-probe combinations were designed based on the target gene sequences (Rhizopus, Mucor, Rhizopus, Rhizopus, and Cladosporium), with each combination required to specifically cover the fungal species as shown in Table 1. In this invention, a total of 14 Mucor species can be detected from the five Mucor genera. The designed primer-probe sequences for Group 1 are shown in Table 2, and the probe labeling is shown in the first column of Tables 8 and 11.

[0030] Table 1. Primers and probes can specifically cover bacterial species.

[0031] Table 2 Primer and probe sequences

[0032] The primer-probe combination 1 designed above was used to verify the specificity within the Mucorales. As shown in Table 3, this primer set has good and strong specificity and does not react specifically with species within the Mucorales. It is unclear whether there is cross-reactivity with other fungi. Therefore, the primer-probe combination designed above will continue to be used to verify the specificity of other fungi.

[0033] Table 3. Results of primer specificity test (CT value) within the Mucorales order.

[0034] 2. Primer specificity testing Genomic DNA from Streptococcus pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Enterobacter faecalis, Escherichia coli, Proteus mirabilis, Aspergillus versicolor, Aspergillus terreus, Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Rhizopus, Mucor, Rhizopus, Rhizopus, Cladosporium, Saccharomyces cerevisiae, Aspergillus nidus, Mycobacterium kansas, Gordoniae, and Lactobacillus acidophilus were collected and subjected to specificity tests with different primers. As shown in Table 4, primer probe set 1 has good specificity and does not cross-react with common human and clinical bacteria and fungi. The finally confirmed primer probe sequences are shown in Table 2.

[0035] Table 4. Results of primer specificity test

[0036] Comparative Example 1 Primer and probe screening (can be used for fluorescent PCR and digital PCR) Another set of primer-probe combinations 2 was designed based on the same target gene sequences (Rhizopus, Mucor, Rhizopus, Rhizopus, and Clematis). Each primer-probe combination 2 was required to specifically cover the fungal species, as shown in Table 1. The fluorescent labeling groups of the probes in combination 2 are the same as those in combination 1 (see the first column of Tables 8 and 11). The sequences of this set of primers-probes are shown in Table 5.

[0037] Table 5 Primer-Probe Combination 2 Sequences

[0038] The specificity of the above-designed primer-probe combination 2 within the Mucorales order was verified. As shown in Table 6, this primer-probe combination has poor specificity and will not be used in subsequent tests.

[0039] Table 6. Results of primer specificity test (CT value) within the Mucorales order.

[0040] Example 2 Preparation of plasmids (including those from the genera *Strombyx*, *Mucor*, *Rhizopus*, *Rhizopus*, *Clostridium*, and internal reference). Construction of cloning plasmids 1) Cloning of the *Strombyx mori* gene fragment: Genomic DNA of *Strombyx mori* was obtained. Using this genomic DNA as a template, PCR amplification was performed using primers DL-1 / DL-2 to obtain a positive control DNA fragment of *Strombyx mori*. The PCR product was recovered and purified, and then inserted into the ECoRI restriction site of the multiple cloning site in the plasmid vector (PUC-T). The product was transformed into *E. coli* DH5α, and the recombinant clones were sequenced for verification. After culturing the recombinant transformants, the plasmid was extracted to obtain the *Strombyx mori* plasmid. The primers were designed by this invention, and the plasmid construction was completed by Shanghai Sangon Biotech Co., Ltd.

[0041] DL-1: 5'-GCATATCAATAAGCGGAGGA-3' (SEQ ID NO. 28); DL-2: 5'-TCGCAGGAACCAGCTACTAG-3' (SEQ ID NO. 29); Inserted DNA sequence (1030 bp) into the plasmid of *Streptomyces*: 2) Cloning of Mucor gene fragments: Genomic DNA of the Mucor genus was obtained. Using this genomic DNA as a template, PCR amplification was performed using primers DL-3 / DL-4 to obtain the Mucor DNA fragment. The PCR product was recovered and purified, and ligated into the ECoRI restriction site of the multiple cloning site in the plasmid vector (PUC-T). The transformed bacteria were 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 Mucor plasmid. The primers were designed by this invention, and the plasmid construction was completed by Shanghai Sangon Biotech Co., Ltd.

[0042] DL-3: 5'-AAGTAAAAGTCGTAACAAGGTTTC-3' (SEQ ID NO. 22); DL-4: 5'-TATGCTTAAGTCAGCGGGTAATCC-3' (SEQ ID NO. 23); Mucor plasmid inserted DNA sequence (643 bp): (SEQ ID NO.24); 3) Cloning of *Rhizopus* gene fragments: Genomic DNA of *Rhizopus* was obtained. Using this genomic DNA as a template, PCR amplification was performed using primers DL-5 / DL-6 to amplify the *Rhizopus* DNA fragment. The PCR product was recovered and purified, and ligated into the ECoRI restriction site of the multiple cloning site in the plasmid vector (PUC-T). The transformed *Rhizopus* DH5α was then transformed, and the recombinant transformants were sequenced for verification. After culturing the recombinant transformants, the plasmid was extracted to obtain the *Rhizopus* plasmid. The primers were designed by this invention, and the plasmid construction was completed by Shanghai Sangon Biotech Co., Ltd.

[0043] DL-5: 5'-TACCTGGTTGATCCTGCCAG-3' (SEQ ID NO. 25); DL-6: 5'-TAGCAGATTAAGGCTCGTT-3' (SEQ ID NO. 26); The DNA sequence inserted into the Rhizopus plasmid (1300 bp): 4) Cloning of Rhizopus gene fragments: Genomic DNA of Rhizopus was obtained. Using this genomic DNA as a template, PCR amplification was performed using primers DL-7 / DL-8 to obtain Rhizopus DNA fragments. The PCR product was recovered and purified, and ligated into the ECoRI restriction site of the multiple cloning site in the plasmid vector (PUC-T). The vector 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 Rhizopus plasmid. The primers were designed by this invention, and the plasmid construction was completed by Shanghai Sangon Biotech Co., Ltd.

[0044] DL-7: 5'-CCTTAGTTTTCCCTCAAGTC-3' (SEQ ID NO. 19); DL-8: 5'-ATCATTTAATTATGTTAAAGC-3' (SEQ ID NO. 20); Rhizopus plasmid inserted DNA sequence (1076 bp): 5) Cloning of the *Clostridium clavatum* gene fragment: Genomic DNA of *Clostridium clavatum* was obtained. Using this genomic DNA as a template, PCR amplification was performed using primers DL-9 / DL-10 to obtain the *Clostridium clavatum* DNA fragment. The PCR product was recovered and purified, and ligated into the ECoRI restriction site of the multiple cloning site in the plasmid vector (PUC-T). The fragment 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 *Clostridium clavatum* plasmid. The primers were designed by this invention, and the plasmid construction was completed by Shanghai Sangon Biotech Co., Ltd.

[0045] DL-9: 5'-CTAGAGCTAATACATGCAGA-3' (SEQ ID NO. 31); DL-10: 5'-GAAAGAGCTTTCAATCTGTC-3' (SEQ ID NO. 32); The plasmid inserted DNA sequence of *Clostridium clethodimense* (1110 bp) is as follows: 6) Cloning of the internal reference gene fragment: The construction process of the internal reference plasmid involves obtaining human genomic DNA, using human genomic DNA as a template, and performing PCR amplification with primers A and B to obtain a human DNA fragment. This human DNA fragment is then ligated into the ECoRI restriction site of the multiple cloning site in the PUC-T vector to construct the internal reference plasmid. The primers were designed by this invention, and the plasmid construction was completed by the Hangzhou branch of Beijing Qingke Biotechnology Co., Ltd.

[0046] Primer A: 5'-ATGAATGGGCAGCCGTTAG-3' (SEQ ID NO.34); Primer B: 5'-CTCAAAGGGCAGGAGTAAA-3' (SEQ ID NO.35); Internal reference plasmid insert DNA fragment sequence (780bp): (SEQ ID NO.36); Example 3 Establishment of multiplex fluorescent PCR and quantitative digital PCR systems 1. Annealing temperature selection Temperature gradient PCR was performed on five primer pairs for Mucorales, Rhizopus, Rhizopus, and Clavicipitolus. The results showed that the overall specificity of the five Mucorales fungi was good when the annealing temperature was around 60℃ and 62℃.

[0047] To further confirm the annealing temperature, three temperatures (60℃, 61℃, and 62℃) were selected for fluorescent PCR to compare their amplification efficiency: when the annealing temperature was 60℃, the amplification efficiency of each target gene was the highest (except for Rhizopus, but 60℃ was selected for all genes to keep the amplification conditions consistent) (Table 7).

[0048] Table 7 Results of annealing temperature experiment for fluorescent PCR

[0049] 2. Establishment of multiplex fluorescent PCR system 2.1 Selection of reaction solution Three reaction solutions (reaction solution 1, reaction solution 2, and reaction solution 3) were selected for multiplex fluorescent PCR amplification of Mucorales. The results showed (see Table 8) that reaction solution 3 had the best performance, and the sensitivity of each target gene and internal reference was higher under the amplification of reaction solution 3 than the other two reaction solutions.

[0050] Reaction solution information: Reaction solution 1: Product name is Anstart Taq DNA Polymerase, catalog number is MD006-01, purchased from Phytobio Biotechnology Co., Ltd.; Reaction solution 2: Product name is PerfectStart® III Fast Probe qPCR SuperMix UDG, catalog number is AQ722-01, purchased from TransGen Biotech Co., Ltd. Reaction solution 3: Product name is Hieff Unicon® Universal TaqMan multiplex qPCRmaster mix (UDG plus), catalog number is 11212ES08, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0051] The fluorescent and quenching groups of various Mucor probes are labeled as follows: Table 8 Selection of Reaction Solution

[0052] 2.2 Investigation into the Concentration of the Mixture To achieve optimal detection sensitivity, probe concentration was optimized (see Table 9).

[0053] Table 9 Optimization of probe concentration

[0054] Probe concentration optimization results: The probe concentration for both detection mixtures 1 and 2 was 0.15 μM. Based on this, the primer concentration was optimized (see Table 10).

[0055] Table 10 Optimization of primer concentration

[0056] Primer optimization results showed that the sensitivity of each target gene and internal reference was optimal when the primer concentration was 0.2 μM for both detection mixtures 1 and 2.

[0057] 3. Establishment of a quantitative digital PCR system 3.1 Selection of reaction solution Three reaction solutions (reaction solution 1, reaction solution 2, and reaction solution 3) were selected for multiplex digital PCR amplification of Mucorales. The results showed (see Table 11) that reaction solution 1 had the best performance. Under the amplification of reaction solution 1, the positive and negative results of each target gene and internal control were normally separated, and the signal-to-noise ratio was appropriate.

[0058] Reaction solution information: Reaction solution 1: Product name is Multiplex qPCR mix, catalog number is DL-D-001, purchased from Hangzhou Dilan Biotechnology Co., Ltd.; Reaction solution 2: Product name is PerfectStart® III Fast Probe qPCR SuperMix UDG, catalog number is AQ722-01, purchased from TransGen Biotech Co., Ltd. Reaction solution 3: Hieff Unicon® Universal TaqMan multiplex qPCR master mix (UDG plus), catalog number 11212ES08, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0059] Table 11 Selection of Reaction Solution

[0060] 3.2 Investigation into the concentration of the mixed solution To optimize the performance of digital PCR detection, primer and probe concentrations were optimized (see Table 12). Optimization results showed that when the primer-probe concentration in the detection mixture was 0.3 μM:0.15 μM, the positive and negative results were clearly distinguished, and the signal-to-noise ratio was suitable.

[0061] Table 12 Investigation of primer and probe concentrations in the mixed solution

[0062] Example 4 Composition of Multiplex Fluorescent PCR and Quantitative Digital PCR Kits 1. Composition of the multiplex fluorescence reagent kit (1) Detection mixture 1: contains primers and probes of the genus *Rhizopus*, primers and probes of the genus *Mucor*, primers and probes of the genus *Rhizopus*, and internal reference primers and probes; the final concentration of each primer is 0.2 μM and the final concentration of each probe is 0.15 μM.

[0063] (2) Detection mixture 2: contains Rhizopus primers and probes; small graminearum primers and probes; internal reference primers and probes; the final concentration of each primer is 0.2 μM, and the final concentration of each probe is 0.15 μM.

[0064] (3) Negative control: Plasmid containing human housekeeper gene fragment (internal reference plasmid), with a final concentration of 1000 copies / μL.

[0065] (4) Positive control: The cloning plasmid containing positive control DNA sequences of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Rhizopus*, and *Clostridium* was mixed with the internal reference plasmid in a certain proportion, and the final concentration of each plasmid was 1000 copies / μL.

[0066] (5) Fluorescent PCR reaction solution: containing dUDG (containing dUTP), Taq enzyme, UDG enzyme, reference fluorescence, MgCl2, buffer, etc., purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 11212ES08.

[0067] Table 13 Components and Concentrations of Multiplex Fluorescent PCR Kit

[0068] 2. Components of a digital PCR kit (1) Detection mixture: Mix the specific primers and probes of the five target genes together, including primers and probes of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Rhizopus*, and *Hymenopterus*. The final concentration of each primer is 0.3 μM and the final concentration of each probe is 0.15 μM.

[0069] (2) Positive control: cloned plasmids containing positive control DNA sequences of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Rhizopus*, and *Clostridium* were mixed in a certain proportion, and the final concentration of each plasmid was 1000 copies / μL.

[0070] (3) Digital PCR enzyme: Taq enzyme, provided by the digital PCR universal kit produced by Hangzhou Dilan Biotechnology Co., Ltd., catalog number: DL-D-001.

[0071] (4) Digital PCR buffer: 3×dPCR Buffer, provided by the Digital PCR Universal Kit produced by Hangzhou Dilan Biotechnology Co., Ltd., catalog number: DL-D-001.

[0072] (5) Nuclease-free water: produced by Hangzhou Dilan Biotechnology Co., Ltd. Product No.: HX22020.

[0073] Table 14 Components and Concentrations of the Multiplex Quantitative Digital PCR Kit

[0074] Example 5 Detection methods of multiplex fluorescent PCR and quantitative digital PCR 1. Detection method of multiplex fluorescent PCR Before using a multiplex fluorescent PCR kit for detection, DNA needs to be extracted from the sample to be tested. The DNA extraction can be performed using a nucleic acid extraction or purification kit (catalog number: DL-A-006) produced by Hangzhou Dilan Biotechnology Co., Ltd.

[0075] 1.1 Preparation of PCR reaction tubes (reagent preparation area) (1) Determine the number of reaction tubes n (number of samples + negative control + positive control); remove test mixture 1 or 2 and PCR reaction solution, and thaw on ice or at room temperature. All kit components need to be briefly centrifuged before use. Each reaction system is shown in Table 15: Table 15 Multiplex Fluorescent PCR Reaction System

[0076] Calculate the amount of each reagent (except for the sample / control) according to the number of reaction tubes n, add them to the centrifuge tube, mix thoroughly, centrifuge briefly, and then aliquot 19 μL into each PCR reaction tube.

[0077] 1.2 Sample addition (sample processing area or sample addition area) Add 6 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, centrifuge briefly, and transfer to the sample detection area.

[0078] 1.3 PCR amplification and fluorescence detection (sample detection area) Place the prepared reaction tubes in a fluorescence PCR instrument and perform the amplification reaction and detection according to the edited sample information under the following conditions (Table 16): Table 16 Fluorescent PCR Amplification Reaction Procedure

[0079] 1.4 Setting of Results Analysis Conditions (1) When analyzing the amplification plot results, set the plot type to: ΔRn vs Cycle.

[0080] (2) Baseline setting: The analysis software of the fluorescence PCR instrument automatically sets the baseline, with the number of cycles from 2 to 3 cycles before the first amplification curve as the baseline.

[0081] (3) Threshold setting: The threshold line of the analysis software of the fluorescence PCR instrument 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.

[0082] 1.5 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: The negative control target gene has no Ct value or is "Undetermined", while the internal control Ct value is <44.00. Positive control: The Ct value of the target gene in the positive control is ≤38.00, while the Ct value of the internal reference is <44.00.

[0083] 1.6 Reading Experimental Results According to the instruction manual for the analysis software 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 Analyse in the software. The system will automatically generate the results, observe the Ct value of the amplification curve for each sample, and download it to an Excel file.

[0084] 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 17.

[0085] Table 17 Method for Determining Ct Value Results

[0086] In the presence of high concentrations of the target gene, its amplification may result in a negative internal reference test result.

[0087] 2. Detection method of multiplex quantitative digital PCR Before using a multiplex quantitative digital PCR kit for testing, DNA needs to be extracted from the sample to be tested. The DNA extraction can be performed using a nucleic acid extraction or purification kit (catalog number: DL-A-006) produced by Hangzhou Dilan Biotechnology Co., Ltd.

[0088] The instruments used in multiplex quantitative digital PCR detection, namely the droplet preparation instrument, PCR amplifier, and chip reader, are all products of the "Qing" series of the BioDigital digital PCR system by Shanghai Xiaohai Turtle Technology Co., Ltd., including three instruments: 1) Fully automatic sample processing system (Hunan Zhuzhou Medical Equipment Preparation 20210021), model Loader Z200; 2) PCR amplifier (Hunan Medical Device Approval 20212220277), model CyclerZ200; 3) Biochip reader (Hunan Medical Device Approval 20212220236), model ImagerZ200.

[0089] 2.1 Preparation of quantitative digital PCR reaction tubes (reagent preparation area) Determine the number of reaction tubes n required (number of samples + negative control + positive control); take out nuclease-free water, digital PCR buffer, and digital PCR enzyme, and thaw them on ice or at room temperature. All kit components need to be centrifuged briefly before use. The reaction system for each sample is shown in Table 18: Table 18 Multiplex digital PCR reaction system

[0090] Calculate the usage amount of each of the above reagents (except for samples / controls) according to the number of reaction tubes n, and add them to a centrifuge tube. Mix well (it is recommended to mix slowly and repeatedly with a pipette while avoiding liquid splashing or generating a large number of bubbles), and after centrifuging briefly, dispense 23 μL into each PCR reaction tube.

[0091] 2.2 Sample addition (sample processing area or sample addition area) Add 7 μL of DNA of the test sample, negative control, or positive control sample to the prepared PCR reaction tubes respectively. After pasting the tube sealing film, centrifuge briefly, transfer them to the corresponding positions of the droplet preparation instrument, open the tube sealing film, and add oil A and oil B in a ratio of 2:1 at the corresponding positions on the oil trough plate (2 + 1 mL can prepare 24 digital PCR chips). After placing the consumables (chips, pipette tips, waste boxes, etc.), the instrument can be started to prepare droplets.

[0092] 2.3 Quantitative digital PCR amplification Place the reaction chip with droplets prepared in the digital PCR amplifier, and perform the amplification reaction according to the following conditions (Table 19) according to the edited sample information: Table 19 Quantitative digital PCR amplification reaction program

[0093] 2.4 Reading of the chip Transfer the amplified chip to a chip reader for reading. Select the FAM, HEX, ROX, CY5, and Quasar705 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.

[0094] 2.5 Quality Control Standards The positive and negative controls of this kit must simultaneously meet the following conditions; otherwise, the experiment will be considered invalid and must 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" effect). The negative control consists of only one layer, and its lower layer is roughly the same as the positive control's lower layer, with no discrete droplets in the upper part.

[0095] 2.6 Data Analysis Open the read chip data using the Little Turtle Biochip Analysis 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 a clear distinction between positive and negative results for the positive control and that all points for the negative control are negative. Adjust the threshold line for the detection group to the same position as the positive and negative control, 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 check for sample addition issues and repeat the experiment to ensure a clear distinction between positive and negative results. After adjusting the thresholds, the "Results List" will display the detection results of the chip. The method for determining the results is shown in Table 20.

[0096] Table 20 Methods for Interpreting Quantitative Digital PCR Results

[0097] Example 6 Performance evaluation of multiplex fluorescent PCR and quantitative digital PCR kits 1. Performance evaluation of the multiplex fluorescent PCR kit 1.1 Detection Limit Using the multiplex fluorescent PCR kit of Example 4 of this invention, and following the multiplex fluorescent PCR method of Example 5, positive reference plasmids of five fungi in the Mucorales order were detected. The detection results are shown in Table 21. Figure 2 The results showed that *Rhizopus*, *Mucor*, *Rhizopus*, *Rhizopus*, and *Clostridium* could be detected in 5 replicates at 100 copies / mL, and the limit of detection for each target gene was 100 copies / mL.

[0098] Table 21 Results of Detection Limit Experiment

[0099] 1.2 Model Equivalence Using the multiplex fluorescent PCR kit of Example 4 of this invention, and following the multiplex fluorescent PCR method of Example 5, the PCR was performed on the company's existing ABI 7500 fluorescent PCR instrument and Hongshi fluorescent PCR instrument. The results show that the ABI 7500 fluorescent PCR instrument and Hongshi fluorescent PCR instrument yielded equivalent results (Table 22).

[0100] Table 22 Model Equivalence Test Data

[0101] 1.3 Real Sample Testing Eight ml of bronchoalveolar lavage fluid sample was provided to this invention by a maternal and child health hospital. DNA was extracted using an extraction kit, and the multiplex fluorescent PCR kit of Example 4 of this invention was used, following the multiplex fluorescent PCR method of Example 5. The Rhizopus genus nucleic acid test was positive, while the nucleic acid tests for Rhizopus, Mucor, Rhizopus, and Cleopatra were negative (see Table 23). The test results are consistent with clinical diagnosis and mNGS sequencing results.

[0102] Table 23 Detection Results of Real Samples

[0103] 2. Performance evaluation of the multiplex quantitative digital PCR kit 2.1 Precision Results Analysis The plasmids at the following two concentrations were tested, with 10 replicates per concentration: 1000 copies / μL and 100 copies / μL. The mean and standard deviation of the test results (concentration values) were calculated for each concentration, and then the CV value was calculated. The judgment criterion was that the CV value should be ≤ 10% at both the high and low concentrations. (Test results are shown in Table 24.) Figure 3 The results showed that the precision CVs for the two concentrations of Rhizopus (FAM) were 3.24% and 4.32%, respectively; for Mucor (HEX) they were 4.70% and 9.70%, respectively; for Rhizopus (ROX) they were 4.71% and 3.16%, respectively; for Cyperus (CY5) they were 4.25% and 7.01%, respectively; and for Quasar705 they were 5.13% and 6.12%, respectively. All of these values ​​were less than 10%, which meets the precision requirements.

[0104] Table 24 Results of 10 runs for each of the two concentrations of the precision reference material (plasmid).

[0105] 2.2 Analysis of Accuracy Results The following four concentrations of plasmid reference standards were tested, with 10 replicates per concentration: high (1000 copies / μL); medium (100 copies / μL); low (10 copies / μL); and very low (1 copy / μL). The deviation between the logarithmic measured concentration and the logarithmic measured concentration was calculated for each concentration. The deviation should be ≤ ±0.45 Log values. The results are shown in Table 25. Figure 4 The deviations between the logarithmic values ​​of the detected concentration and the logarithmic values ​​of the theoretical concentration are all within ±0.45 Log values, which meets the accuracy verification requirements.

[0106] Table 25: Results of 10 trials for each of the four concentrations of the accuracy reference material (plasmid).

[0107] 2.3 Exploration and Confirmation of Linear Range Take a tube of high-concentration simulated nucleic acid sample and serially dilute it 4-fold to test a total of 7 concentrations (unit: copies / μL): 1.60E+04; 4.00E+03; 1.00E+03; 2.50E+02; 6.25E+01; 1.56E+01; 3.91E+0. Each concentration was tested three times (Table 26). Calculate the logarithmic correlation coefficient between the theoretical concentration and the average of the measured concentrations. R0 is required. 2 >0.980. The correlation analysis results are shown in Table 27. The measured values ​​of *Rhizopus*, *Mucor*, *Rhizopus*, *Strombyx*, and *Clostridium* were significantly positively correlated with the expected theoretical values; all R values... 2 The value >0.980 and p <0.0001 indicate that the linear range of the reagent meets the validation criteria.

[0108] Table 26 Experimental data on linear range

[0109] Table 27 Statistical table of regression analysis of the logarithmic values ​​of the mean theoretical concentration and the mean measured concentration for each channel.

[0110] 2.4 Detection Limit Confirmation Plasmids from the genera *Rhizopus*, *Pheromus*, *Mucor*, *Rhizopus*, and *Clostridium* were diluted to a concentration of 0.3 copies / μL as reference standards for the detection limit. Five repeated tests were performed at the set lowest detection limit concentration and lower concentration values, and the concentrations (copies / μL) were recorded.

[0111] When the number of positive points in the FAM / ROX / HEX / CY5 / Quasar705 channels is ≥1, it is considered a positive result for each channel. A detection rate of 5 / 5 is considered acceptable. The test results are shown in Table 28. Figure 5 Five repeated tests were performed at the set minimum detection limit concentration. All channels detected the virus, and the number of positive points was ≥ 1 for each channel. The results were all positive, which met the requirements of the detection limit setting.

[0112] Table 28: Results of 5 tests for the lowest detection limit reference sample (plasmid)

[0113] 2.5 Real Sample Detection Eight mL of bronchoalveolar lavage fluid was provided to this invention by a maternal and child health hospital. DNA was extracted using the extraction kit of this invention. After testing with a multiplex digital PCR kit, the nucleic acid test for Rhizopus was positive, while the nucleic acid tests for Rhizopus, Mucor, Rhizopus, and Cleopatra were negative. The test results were consistent with the clinical diagnosis and mNGS sequencing results.

[0114] Based on the above experimental data, the outstanding advantages of the primer-probe set and kit of this invention can be summarized as follows: First, the design of the primer and probe sets for the five Mucor genera achieves high specificity and sensitivity. Using the five sets of primers and probes together not only avoids cross-reactivity among the five Mucor genera, but also avoids cross-reactivity with other common fungi and bacteria. Given the high homology of the genomic sequences of various Mucor genera, this invention enables the development of methodologically specific primers and probes unique to each Mucor genera, giving it a leading edge in molecular biology nucleic acid detection technology. Second, all five sets of Mucor primers and probes can be used simultaneously for target detection in both multiplex fluorescent PCR and multiplex quantitative digital PCR methods, demonstrating high specificity and sensitivity. The multiplex fluorescent PCR method achieves a detection limit of 100 copies / mL, while the multiplex quantitative digital PCR method achieves a detection limit of 0.3 copies / μL. Furthermore, the five-parameter multiplex fluorescent PCR nucleic acid detection kit and multiplex quantitative digital PCR kit of this invention have achieved industrialization and practical high-efficiency application in various performance indicators such as specificity, detection limit, precision, accuracy, and detection of real clinical samples. In summary, the accurate qualitative and direct quantitative detection of five Mucor genera that can be achieved and implemented by this invention has significant practical value for timely, efficient, and accurate detection of up to five Mucor genera, including fourteen species of Mucor.

[0115] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer and probe set for the detection of fungi in the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Pheretima*, and *Clostridium* by multiplex fluorescent PCR and / or multiplex quantitative digital PCR, characterized in that... The kit includes primers dl-RHI-F1, dl-RHI-R1 and probe dl-RHI-P1 for detecting Rhizopus fungi; primers dl-MUC-F1, dl-MUC-R1 and probe dl-MUC-P1 for detecting Mucor fungi; primers dl-RHIZO-F1, dl-RHIZO-R1 and probe dl-RHIZO-P1 for detecting Rhizopus fungi; primers dl-ABS-F1, dl-ABS-R1 and probe dl-ABS-P1 for detecting Rhizopus fungi; and primers dl-CUN-F1, dl-CUN-R1 and probe dl-CUN-P1 for detecting Cladosporium fungi. The nucleotide sequence of dl-RHI-F1 is shown in SEQ ID NO.1; the nucleotide sequence of dl-RHI-R1 is shown in SEQ ID NO.2; and the nucleotide sequence of dl-RHI-P1 is shown in SEQ ID NO.

2. The nucleotide sequence of dl-MUC-F1 is shown in SEQ ID NO. 3; the nucleotide sequence of dl-MUC-R1 is shown in SEQ ID NO. 5; the nucleotide sequence of dl-MUC-P1 is shown in SEQ ID NO. 6; the nucleotide sequence of dl-RHIZO-F1 is shown in SEQ ID NO. 7; the nucleotide sequence of dl-RHIZO-R1 is shown in SEQ ID NO. 8; the nucleotide sequence of dl-RHIZO-P1 is shown in SEQ ID NO. 9; the nucleotide sequence of dl-ABS-F1 is shown in SEQ ID NO. 10; the nucleotide sequence of dl-ABS-R1 is shown in SEQ ID NO. 11; the nucleotide sequence of dl-ABS-P1 is shown in SEQ ID NO. 12; the nucleotide sequence of dl-CUN-F1 is shown in SEQ ID NO. 13; the nucleotide sequence of dl-CUN-R1 is shown in SEQ ID NO.

14. As shown in NO.14; the nucleotide sequence of dl-CUN-P1 is shown in SEQ ID NO.15; the 5' ends of dl-RHI-P1, dl-MUC-P1, dl-RHIZO-P1, dl-ABS-P1 and dl-CUN-P1 are labeled with fluorescent groups, and the 3' ends are labeled with quenching groups.

2. The primer-probe set according to claim 1, characterized in that, The primer-probe set further includes internal reference primers dl-GH-F1 and dl-GH-R1 and probe dl-GH-P1; the nucleotide sequence of dl-GH-F1 is shown in SEQ ID NO.16; the nucleotide sequence of dl-GH-R1 is shown in SEQ ID NO.17; the nucleotide sequence of dl-GH-P1 is shown in SEQ ID NO.18; the 5' end of dl-GH-P1 is labeled with a fluorescent group, and the 3' end is labeled with a quencher group.

3. The primer-probe set according to claim 1 or 2, characterized in that, The fluorescent group is selected from any one of FAM, ROX, Texas-Red, HEX, VIC, JOE, TET, CY5, Quasar705, CY5.5, ATTO425, and AF405; the quenching group is selected from any one of BHQ1, BHQ2, BHQ3, and MGB.

4. The use of the primer and probe set according to any one of claims 1 to 3 in the preparation of kits for multiplex fluorescent PCR and / or multiplex quantitative digital PCR to detect fungi of the genera Rhizopus, Mucor, Rhizopus, Rhizopus and Clefylvatica, or kits for detecting infectious diseases caused by fungi of the genera Rhizopus, Mucor, Rhizopus, Rhizopus and Clefylvatica.

5. A kit for detecting fungi of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Pheretima*, and *Clostridium*, characterized in that: It includes the primer and probe set and reaction solution as described in any one of claims 1 to 3.

6. The reagent kit according to claim 5, characterized in that, The reaction solution includes multiplex fluorescent PCR reaction solution and / or multiplex quantitative digital PCR reaction solution.

7. The reagent kit according to claim 5, characterized in that, The kit also includes a multiplex fluorescent PCR positive control and a multiplex fluorescent PCR negative control, and / or the kit also includes a multiplex quantitative digital PCR reaction solution positive control.

8. The reagent kit according to claim 7, characterized in that, The multiplex fluorescent PCR positive control includes cloning plasmids and internal reference plasmids containing nucleotide sequences of fungal genes from the gene gene *Rhizopus*, *Mucor*, *Rhizopus*, *Pterygota*, and *Clostridium*, respectively; the multiplex fluorescent PCR negative control includes an internal reference plasmid; the multiplex quantitative digital PCR reaction solution positive control includes cloning plasmids containing nucleotide sequences of fungal genes from the gene ...

9. The reagent kit according to claim 8, characterized in that, The cloning plasmids containing gene nucleotide sequences of Rhizopus, Mucor, Rhizopus, Rhizopus, and Cladosporium hainanense fungi respectively include cloning plasmids containing Rhizopus gene nucleotide sequences as shown in SEQ ID NO.21, Rhizopus gene nucleotide sequences as shown in SEQ ID NO.24, Rhizopus gene nucleotide sequences as shown in SEQ ID NO.27, Rhizopus gene nucleotide sequences as shown in SEQ ID NO.30, and Cladosporium hainanense gene nucleotide sequences as shown in SEQ ID NO.33; the internal reference plasmid contains the human housekeeper gene nucleotide sequence as shown in SEQ ID NO.

36.

10. The kit according to claim 4, characterized in that, When using multiplex fluorescent PCR to detect fungi of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Phyllostachys*, and *Clostridium*, the primer concentration was 0.2 μM and the probe concentration was 0.15 μM. When using multiplex quantitative digital PCR to detect fungi of the genera *Rhizopus*, *Mucor*, *Rhizopus*, *Phyllostachys*, and *Clostridium*, the primer concentration was 0.3 μM and the probe concentration was 0.15 μM.

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