Nucleic acid combination product, kit for detecting pathogenic fungi and detection method

Abstract

The invention relates to a nucleic acid combination product, a kit for detecting pathogenic fungi and a detection method. The nucleic acid combination product provided by the invention comprises a composition of a plurality of groups of primer pairs and detection probes, is wide in detection coverage and high in inclusiveness, can simultaneously detect a plurality of pathogenic fungi in a single-tube reaction system, has relatively high sensitivity and specificity, is a pathogenic fungi detection product with relatively comprehensive detection targets at present, and has a wide application prospect. And an auxiliary diagnosis basis is provided for clinical examination.

Description

Technical Field

[0001] This application belongs to the technical field of pathogenic microorganism detection, and particularly relates to a nucleic acid combination product, a kit for detecting pathogenic fungi, and a detection method. Background Art

[0002] Human pathogenic pathogens are mainly divided into viruses, bacteria, fungi, etc. Among them, fungi are one of the common flora in bacterial infections and nosocomial infections. In recent years, with the occurrence of various malignant diseases, chronic consumptive diseases, and immunodeficiency diseases, the extensive use of broad-spectrum antibiotics, glucocorticoids, immunosuppressants, and interventional therapies has led to an obvious increasing trend in the fungal infection rate.

[0003] The lungs are the most frequently invaded site of fungal infection, accounting for 50-60% of all visceral fungal infections. Invasive pulmonary fungal infection (IPFI) refers to a disease caused by acute and chronic histopathological damage directly caused by fungi invading (non-parasitic, allergic or toxin poisoning) the lungs or bronchi. It is also one of the respiratory diseases that seriously threaten public health worldwide and is divided into two types: primary and secondary. Primary IPFI is caused by pathogenic fungi; while secondary IPFI is caused by opportunistic pathogenic fungi and is more common and highly regarded in clinical practice. The common fungi causing IPFI are mainly Candida, Aspergillus, Cryptococcus, Zygomycetes (mainly referring to Mucor), and Sporothrix, etc.

[0004] The incidence of IPFI shows an increasing trend year by year, and a considerable number of patients belong to secondary infections of end-stage diseases. Due to the high mortality rate, it is also called "terminal infection". Most pulmonary fungal infections are secondary to high-risk groups with immune function deficiencies such as malignant tumors, blood diseases, respiratory diseases, AIDS, and severe liver diseases. Since the clinical manifestations of IPFI are often non-specific, early diagnosis is difficult, and the condition is easily masked by the primary disease, which is likely to cause misdiagnosis and missed diagnosis, delaying treatment. The mortality rate of patients with pulmonary fungal infection without timely treatment is as high as 30% - 80%.

[0005] Traditional pathogenic fungal diagnostic methods mainly include direct microscopic examination of fungi, fungal culture and identification, and fungal serological examination. However, these methods all have the problem of low sensitivity, and it is also difficult to simultaneously screen for multiple pathogens. The PCR methodology directly detects pathogen nucleic acids, with high sensitivity, good specificity, simplicity, and rapidity, and it is currently the fastest-developing and most widely applied pathogen detection method. Currently, there are relatively few PCR detection kits for pathogenic fungi on the market, with single-detection and triple-detection products. However, there are diverse common pathogenic fungal species, so for the current demand for more pathogen detections, more tubes of reagents are needed to achieve this, bringing higher costs and inconvenience to clinical testing. Therefore, there is an urgent need to develop a fungal nucleic acid multiplex detection kit that can react in the same tube, is convenient and fast to operate, and has high sensitivity and specificity. Summary of the Invention

[0006] Based on this, an embodiment of the present application provides a nucleic acid combination product, a kit for detecting pathogenic fungi, and a detection method.

[0007] One aspect of the present application provides a nucleic acid combination product, which includes multiple groups of the following probe primer combinations:

[0008] (1) A primer pair with nucleotide sequences as shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence as shown in SEQ ID NO:3, and a PCO probe with a nucleotide sequence as shown in SEQ ID NO:4;

[0009] (2) A primer pair with nucleotide sequences as shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence as shown in SEQ ID NO:5, and a PCO probe with a nucleotide sequence as shown in SEQ ID NO:6;

[0010] (3) A primer pair with nucleotide sequences as shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence as shown in SEQ ID NO:7, and a PCO probe with a nucleotide sequence as shown in SEQ ID NO:8;

[0011] (4) A primer pair with nucleotide sequences as shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence as shown in SEQ ID NO:9, and a PCO probe with a nucleotide sequence as shown in SEQ ID NO:10;

[0012] (5) A primer pair with nucleotide sequences as shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence as shown in SEQ ID NO:11, and a PCO probe with a nucleotide sequence as shown in SEQ ID NO:12;

[0013] Primer pairs with nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence shown in SEQ ID NO:13, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:14;

[0014] Primer pairs with nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence shown in SEQ ID NO:15, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:16;

[0015] Primer pairs with nucleotide sequences shown in SEQ ID NO:17 to SEQ ID NO:18, a THO probe with a nucleotide sequence shown in SEQ ID NO:19, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:20;

[0016] Primer pairs with nucleotide sequences shown in SEQ ID NO:21 to SEQ ID NO:22, a THO probe with a nucleotide sequence shown in SEQ ID NO:23, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:24;

[0017] Primer pairs with nucleotide sequences shown in SEQ ID NO:25 to SEQ ID NO:26, a THO probe with a nucleotide sequence shown in SEQ ID NO:27, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:28;

[0018] Primer pairs with nucleotide sequences shown in SEQ ID NO:29 to SEQ ID NO:30, a THO probe with a nucleotide sequence shown in SEQ ID NO:31, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:32;

[0019] Primer pairs with nucleotide sequences shown in SEQ ID NO:29 to SEQ ID NO:30, a THO probe with a nucleotide sequence shown in SEQ ID NO:33, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:34;

[0020] Primer pairs with nucleotide sequences shown in SEQ ID NO:29 to SEQ ID NO:30, a THO probe with a nucleotide sequence shown in SEQ ID NO:35, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:36;

[0021] A primer pair with nucleotide sequences as shown in SEQ ID NO: 29 to SEQ ID NO: 30, a THO probe with a nucleotide sequence as shown in SEQ ID NO: 37, and a PCO probe with a nucleotide sequence as shown in SEQ ID NO: 38.

[0022] In some embodiments, the nucleic acid combination product includes primers and probes with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 38.

[0023] In some embodiments, the 3'-end of the PCO detection probe is labeled with a phosphate group; both ends of the THO detection probe are respectively labeled with a fluorescent reporter group and a fluorescent quenching group;

[0024] Optionally, the 5'-end of the THO detection probe is labeled with the fluorescent reporter group, and the 3'-end is labeled with the fluorescent quenching group;

[0025] Optionally, the fluorescent reporter group includes one or more of FAM, VIC, ROX, and CY5;

[0026] Optionally, the fluorescent quenching group includes one or more of BHQ1 and BHQ2.

[0027] In some embodiments, the nucleic acid combination product further includes an internal reference gene detection primer pair or / and an internal reference gene detection probe;

[0028] Optionally, the internal reference gene detection primer pair includes a forward primer with a nucleotide sequence as shown in SEQ ID NO. 39 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO. 40;

[0029] Optionally, the nucleotide sequence of the internal reference gene detection probe is as shown in SEQ ID NO. 41.

[0030] On the other hand, the present application provides a kit for detecting pathogenic fungi, which includes: the above-mentioned nucleic acid combination product.

[0031] In some embodiments, the kit further includes: one or more of a nucleic acid release reagent, a nucleic acid extraction reagent, and a nucleic acid amplification reagent;

[0032] Optionally, the nucleic acid amplification reagent includes one or more of DNA polymerase, dNTPs, UNG enzyme, PCR buffer, and Mg2+.

[0033] In some embodiments, the kit further includes one or more of a positive control product and a negative control product.

[0034] In some of these embodiments, the positive control product includes one or more of a Candida albicans control product, a Cryptococcus control product, and an Aspergillus fumigatus control product;

[0035] Or / and, the negative control product includes enzyme-free water.

[0036] Another aspect of the present application provides a method for detecting pathogenic fungi, including:

[0037] Providing a nucleic acid sample to be tested, performing a PCR amplification reaction on the nucleic acid sample to be tested using the above nucleic acid combination product or the above kit, analyzing the obtained amplification product by the melting curve method, and determining the situation of the nucleic acid sample to be tested containing pathogenic fungi according to the obtained analysis result;

[0038] Optionally, the PCR amplification reaction includes an MPA multiplex fluorescence PCR amplification reaction.

[0039] In some of these embodiments, the source of the nucleic acid sample to be tested includes a sputum sample.

[0040] In summary, the present application provides a multiplex fluorescence PCR detection kit for multiplex detection of fungal nucleic acids and its detection method, which can perform typing detection of multiple pathogenic fungi in a single tube. It is simple and fast to operate, compatible with conventional fluorescence quantitative PCR platforms, and can be applied to assist clinicians in diagnosing the pathogen types of fungal infections, which is of great significance for the prevention, control and treatment of diseases such as invasive pulmonary fungal infection (IPFI). BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

[0042] Figure 1 For the amplification curve and melting curve characteristic map results of Candida tropicalis in the FAM channel in Example 2, where the left figure is the amplification curve, the abscissa is the number of cycles, and the ordinate Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the abscissa is the temperature, and the ordinate -d(Rn) / dT is the negative first derivative of the fluorescence signal intensity with respect to the temperature, which is used to more clearly show the rate of change of the fluorescence signal with temperature and can help determine the melting temperature (Tm value) of the product in the melting curve analysis;

[0043] Figure 2Results of the amplification curve and melting curve characteristic spectra of Candida albicans in FAM channel in Example 2. The left figure is the amplification curve, with the abscissa being the number of cycles and the ordinate Rn referring to the fluorescence signal intensity; the right figure is the melting curve, with the abscissa being the temperature and the ordinate -d(Rn) / dT, which is the negative first derivative of the fluorescence signal intensity with respect to the temperature.

[0044] Figure 3 Results of the amplification curve and melting curve characteristic spectra of Candida glabrata in FAM channel in Example 2. The left figure is the amplification curve, with the abscissa being the number of cycles and the ordinate Rn referring to the fluorescence signal intensity; the right figure is the melting curve, with the abscissa being the temperature and the ordinate -d(Rn) / dT, which is the negative first derivative of the fluorescence signal intensity with respect to the temperature.

[0045] Figure 4 Results of the amplification curve and melting curve characteristic spectra of Candida krusei in FAM channel in Example 2. The left figure is the amplification curve, with the abscissa being the number of cycles and the ordinate Rn referring to the fluorescence signal intensity; the right figure is the melting curve, with the abscissa being the temperature and the ordinate -d(Rn) / dT, which is the negative first derivative of the fluorescence signal intensity with respect to the temperature.

[0046] Figure 5 Results of the amplification curve and melting curve characteristic spectra of Candida lusitaniae in FAM channel in Example 2. The left figure is the amplification curve, with the abscissa being the number of cycles and the ordinate Rn referring to the fluorescence signal intensity; the right figure is the melting curve, with the abscissa being the temperature and the ordinate -d(Rn) / dT, which is the negative first derivative of the fluorescence signal intensity with respect to the temperature.

[0047] Figure 6 Results of the amplification curve and melting curve characteristic spectra of Candida auris in VIC channel in Example 2. The left figure is the amplification curve, with the abscissa being the number of cycles and the ordinate Rn referring to the fluorescence signal intensity; the right figure is the melting curve, with the abscissa being the temperature and the ordinate -d(Rn) / dT, which is the negative first derivative of the fluorescence signal intensity with respect to the temperature.

[0048] Figure 7 Results of the amplification curve and melting curve characteristic spectra of Cryptococcus neoformans in VIC channel in Example 2. The left figure is the amplification curve, with the abscissa being the number of cycles and the ordinate Rn referring to the fluorescence signal intensity; the right figure is the melting curve, with the abscissa being the temperature and the ordinate -d(Rn) / dT, which is the negative first derivative of the fluorescence signal intensity with respect to the temperature.

[0049] Figure 8 Results of the amplification curve and melting curve characteristic spectra of Pneumocystis jirovecii in VIC channel in Example 2. The left figure is the amplification curve, with the abscissa being the number of cycles and the ordinate Rn referring to the fluorescence signal intensity; the right figure is the melting curve, with the abscissa being the temperature and the ordinate -d(Rn) / dT, which is the negative first derivative of the fluorescence signal intensity with respect to the temperature.

[0050] Figure 9 The amplification curve and melting curve characteristic spectrum results of the Candida parapsilosis group in the VIC channel in Example 2, wherein the left figure is the amplification curve, the abscissa is the cycle number, and the ordinate Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the abscissa is the temperature, and the ordinate -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity to the temperature;

[0051] Figure 10 The amplification curve and melting curve characteristic spectrum results of the ROX channel Aspergillus flavus complex in Example 2, wherein the left figure is the amplification curve, the abscissa is the cycle number, and the ordinate Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the abscissa is the temperature, and the ordinate -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity to the temperature;

[0052] Figure 11 The amplification curve and melting curve characteristic spectrum results of the pathogenic bacteria of ROX channel mucormycosis in Example 2, wherein the left figure is the amplification curve, the abscissa is the cycle number, and the ordinate Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the abscissa is the temperature, and the ordinate -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity to the temperature;

[0053] Figure 12 The amplification curve and melting curve characteristic spectrum results of the ROX channel Aspergillus terreus complex in Example 2, wherein the left figure is the amplification curve, the abscissa is the cycle number, and the ordinate Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the abscissa is the temperature, and the ordinate -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity to the temperature;

[0054] Figure 13 The amplification curve and melting curve characteristic spectrum results of the ROX channel Aspergillus fumigatus complex in Example 2, wherein the left figure is the amplification curve, the abscissa is the cycle number, and the ordinate Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the abscissa is the temperature, and the ordinate -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity to the temperature;

[0055] Figure 14 The amplification curve and melting curve characteristic spectrum results of the ROX channel Aspergillus niger complex in Example 2, wherein the left figure is the amplification curve, the abscissa is the cycle number, and the ordinate Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the abscissa is the temperature, and the ordinate -d(Rn) / dT is the negative first-order derivative of the fluorescence signal intensity to the temperature;

[0056] Figure 15 The amplification curve and melting curve characteristic spectrum results of 10 negative samples in Example 3, wherein the left figure is the amplification curve and the right figure is the melting curve; Figure 1 The curve is the FAM channel, rightFigure 2 The curve is the VIC channel, on the right Figure 3 The curve is the ROX channel;

[0057] Figure 16 They are the amplification curve and melting curve characteristic atlas results of various common Mucor mycosis pathogens in Example 4. Among them, the left figure is the amplification curve, the abscissa is the number of cycles, and the ordinate Rn refers to the fluorescence signal intensity; the right figure is the melting curve, the abscissa is the temperature, and the ordinate -d(Rn) / dT is the negative first derivative of the fluorescence signal intensity with respect to temperature. Specific embodiments

[0058] The present application will be further described in detail below in combination with the embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosed content of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0059] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs.

[0060] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0061] As used herein, the alternative scopes of the terms "and / or", "or / and", and "and / or" include any one of two or more related listed items, and also include any and all combinations of the related listed items. The aforesaid any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and undoubtedly also includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example is the technical solution of "A, and / or, B, and / or, C, and / or, D", which includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the combination of the four items A, B, C, and D (that is, the technical solution connected by "logical AND").

[0062] In this application, terms such as "multiple", "diverse", "multiple times", and "pluralistic" refer to a quantity greater than or equal to 2 if not otherwise specified. For example, "one or more" means one or greater than or equal to two.

[0063] As used herein, "its combination", "any combination thereof", "any combination mode thereof", etc. include all suitable combination modes of any two or more than two of the listed items.

[0064] In this text, the "suitable" in "suitable combination mode", "suitable mode", "any suitable mode", etc. is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0065] In this application, terms such as "further", "even further", and "especially" are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the protection scope of this application.

[0066] In this application, "optionally", "optional", and "option" mean that it can be either present or absent, that is, it refers to any one of two parallel solutions of "present" or "absent". If "optional" appears multiple times in a technical solution, without special instructions and without contradictions or mutual restrictions, each "optional" is independent of each other.

[0067] In this application, for the technical features described in an open-ended manner, it includes the closed technical solutions composed of the listed features, and also includes the open technical solutions containing the listed features.

[0068] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the selectable numerical values are considered continuous within the above numerical intervals, and include the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, when t is an integer selected from 1 to 10, it means that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0069] In this application, the temperature parameter, unless otherwise specified, allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0070] In this application, %(w / w) and wt% both represent weight percentages, %(v / v) refers to volume percentages, and %(w / v) refers to mass-volume percentages.

[0071] On the one hand, this application provides a nucleic acid combination product, and the above nucleic acid combination product includes multiple groups among the following probe primer combinations:

[0072] (1) A primer pair with a nucleotide sequence as shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence as shown in SEQ ID NO:3, and a PCO probe with a nucleotide sequence as shown in SEQ ID NO:4;

[0073] (2) A primer pair with a nucleotide sequence as shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence as shown in SEQ ID NO:5, and a PCO probe with a nucleotide sequence as shown in SEQ ID NO:6;

[0074] (3) A primer pair with a nucleotide sequence as shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence as shown in SEQ ID NO:7, and a PCO probe with a nucleotide sequence as shown in SEQ ID NO:8;

[0075] (4) primer pairs with nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence shown in SEQ ID NO:9, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:10;

[0076] (5) primer pairs with nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence shown in SEQ ID NO:11, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:12;

[0077] (6) primer pairs with nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence shown in SEQ ID NO:13, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:14;

[0078] (7) primer pairs with nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:2, a THO probe with a nucleotide sequence shown in SEQ ID NO:15, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:16;

[0079] (8) primer pairs with nucleotide sequences shown in SEQ ID NO:17 to SEQ ID NO:18, a THO probe with a nucleotide sequence shown in SEQ ID NO:19, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:20;

[0080] (9) primer pairs with nucleotide sequences shown in SEQ ID NO:21 to SEQ ID NO:22, a THO probe with a nucleotide sequence shown in SEQ ID NO:23, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:24;

[0081] (10) primer pairs with nucleotide sequences shown in SEQ ID NO:25 to SEQ ID NO:26, a THO probe with a nucleotide sequence shown in SEQ ID NO:27, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:28;

[0082] (11) primer pairs with nucleotide sequences shown in SEQ ID NO:29 to SEQ ID NO:30, a THO probe with a nucleotide sequence shown in SEQ ID NO:31, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:32;

[0083] A primer pair with nucleotide sequences shown in SEQ ID NO:29 to SEQ ID NO:30, a THO probe with a nucleotide sequence shown in SEQ ID NO:33, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:34;

[0084] A primer pair with nucleotide sequences shown in SEQ ID NO:29 to SEQ ID NO:30, a THO probe with a nucleotide sequence shown in SEQ ID NO:35, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:36;

[0085] A primer pair with nucleotide sequences shown in SEQ ID NO:29 to SEQ ID NO:30, a THO probe with a nucleotide sequence shown in SEQ ID NO:37, and a PCO probe with a nucleotide sequence shown in SEQ ID NO:38.

[0086] The nucleic acid combination product provided by the present application has a wide detection coverage, can simultaneously detect multiple pathogenic fungi in a single-tube reaction system, and has high sensitivity and specificity.

[0087] In some of these embodiments, the above nucleic acid combination product includes primers and probes with nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:38.

[0088] It is understandable that this application uses the Multiplex Probe Amplification (MPA) technology to develop a 14-pathogen fungal detection kit with melting curve analysis as the typing method, covering a comprehensive range of pathogenic fungi and enabling rapid single-tube detection of pathogen types. The principle of the MPA technology is as follows: For each target, a pair of primers, a fluorescently labeled probe (THO), and a hybridization probe (PCO) that is reverse complementary to the THO probe and incorporates a mismatched base are provided. After the PCR amplification is completed, during the process of increasing the temperature for melting curve analysis (such as from 25°C to 75°C), the THO-PCO hybrid double-strand undergoes a process of fluorescence from presence to absence, thus forming a melting curve with a unique Tm (Melting Temperature). According to the degree of mismatch introduced by the THO-PCO hybrid double-strand and the different mismatched bases, each fluorescence channel can accommodate the melting curve peaks of 4 to 5 targets, and the adjacent melting curve peaks differ by at least 4°C. When there is no target gene in the reaction system, such as in the negative control sample, the THO probe will not be cleaved by Taq polymerase during the amplification stage and will form a melting peak with the PCO during the melting curve stage. When there is a target gene in the reaction system, the THO probe will be cleaved by Taq polymerase during the amplification stage. The higher the target concentration, the more the THO probe will be consumed, and even completely consumed. When performing melting curve analysis, the height of the melting peak of THO and PCO will decrease or disappear. By comparing and analyzing with the melting curve of the negative control sample where THO is not consumed, the type of target present in the sample can be determined, thereby achieving qualitative and typing detection of different pathogens in the sample.

[0089] In some embodiments, the 3'-end of the above-mentioned PCO detection probe is labeled with a phosphate group; the two ends of the above-mentioned THO detection probe are respectively labeled with a fluorescent reporter group and a fluorescent quenching group.

[0090] In some embodiments, the 5'-end of the above-mentioned THO detection probe is labeled with the above-mentioned fluorescent reporter group, and the 3'-end is labeled with the above-mentioned fluorescent quenching group.

[0091] In some embodiments, the above-mentioned fluorescent reporter group includes one or more of FAM, VIC, ROX, and CY5. It can be understood that the absorbance values of these groups are not close to each other, and different channels can be selected, so they will not interfere with each other.

[0092] In some embodiments, the above-mentioned fluorescent quenching group includes one or more of BHQ1 and BHQ2.

[0093] In some embodiments, the present application adopts the MPA multiplex fluorescence probe PCR technology, which can simultaneously detect 14 pathogenic fungi and perform typing through melting curve analysis. The 14 pathogenic fungi include: Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida metapsilosis, Candida orthopsilosis, Candida tropicalis, Candida lusitaniae, Candida auris, Aspergillus Section Fumigati, Aspergillus Section Flavi, Aspergillus Section Nigri, Aspergillus Section Terrei, Mucorales, Cryptococcus neoformans, Cryptococcus gattii, and Pneumocystis jirovecii.

[0094] In some embodiments, the fluorescence reporter group of the THO detection probes for Candida tropicalis, Candida albicans, Candida glabrata, Candida krusei, and Candida lusitaniae is FAM, and the fluorescence quenching group is BHQ1; the fluorescence reporter group of the THO detection probes for Candida auris, Candida parapsilosis complex, Cryptococcus neoformans, and Pneumocystis jirovecii is VIC, and the fluorescence quenching group is BHQ1; the fluorescence reporter group of the THO detection probes for Mucorales, Aspergillus Section Fumigati, Aspergillus Section Flavi, Aspergillus Section Nigri, and Aspergillus Section Terrei is ROX, and the fluorescence quenching group is BHQ2.

[0095] In some embodiments, the above nucleic acid combination product further includes an internal reference gene detection primer pair and / or an internal reference gene detection probe.

[0096] In some embodiments, the above internal reference gene detection primer pair includes a forward primer with a nucleotide sequence as shown in SEQ ID NO. 39 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO. 40.

[0097] In some embodiments, the nucleotide sequence of the above internal reference gene detection probe is as shown in SEQ ID NO. 41.

[0098] The specific sequence is shown in Table 1 below.

[0099] Table 1

[0100]

[0101]

[0102] Another embodiment of the present application provides a kit for detecting pathogenic fungi, and the kit includes the above nucleic acid combination product.

[0103] In some embodiments, the kit further includes one or more of: a nucleic acid release reagent, a nucleic acid extraction reagent, and a nucleic acid amplification reagent.

[0104] In some embodiments, the nucleic acid amplification reagent includes one or more of DNA polymerase, dNTPs, UNG enzyme, PCR buffer, and Mg 2+ among others.

[0105] In some embodiments, the kit further includes one or more of a positive control product and a negative control product.

[0106] In some embodiments, the positive control product includes one or more of a Candida albicans control product, a Cryptococcus control product, and an Aspergillus fumigatus control product.

[0107] In some embodiments, the negative control product includes enzyme-free water.

[0108] Another embodiment of the present application further provides a method for detecting pathogenic fungi, including:

[0109] Providing a nucleic acid sample to be tested, performing a PCR amplification reaction on the nucleic acid sample to be tested using the above nucleic acid combination product or the above kit, analyzing the obtained amplification product by the melting curve method, and determining the presence of pathogenic fungi in the nucleic acid sample to be tested according to the obtained analysis result.

[0110] In some embodiments, the PCR amplification reaction includes an MPA multiplex fluorescence PCR amplification reaction.

[0111] In some embodiments, the source of the nucleic acid sample to be tested includes a sputum sample.

[0112] In some embodiments, the step of analyzing the reaction result includes: respectively obtaining the Ct value and the melting curve characteristic map amplified by the nucleic acid combination product, and determining the result according to the Ct value and the melting curve characteristic map.

[0113] The above-mentioned multiplex fluorescence PCR detection kit for multiplex detection of fungal nucleic acids and its detection method can perform typing detection of 14 pathogenic fungi in a single tube. It is simple and fast to operate, compatible with all conventional fluorescence quantitative PCR platforms, and can be used to assist clinicians in diagnosing the pathogen types of fungal infections, which is of great significance for the prevention, control and treatment of diseases such as invasive pulmonary fungal infection (IPFI).

[0114] In addition, due to the high polymorphism of pathogenic fungi, it can also be used in the research of fields such as sociology (such as species distribution, etc.). This detection method includes detection methods related to disease diagnosis and detection methods not related to disease diagnosis.

[0115] (1) By using the MPA multiplex fluorescence probe PCR technology, typing detection of 4-5 fungal targets can be achieved in a single-color channel. Therefore, 14 fungi can be jointly detected, increasing the detection throughput of the existing fluorescence PCR technology and breaking through the bottleneck that the current human fungal nucleic acid detection kits on the market can only detect 3-4 targets at a time.

[0116] (2) The typing of 14 target fungi depends on the melting curve analysis after PCR amplification. The whole detection process is carried out under single-tube closed conditions, avoiding false positives caused by cross-contamination between samples and environmental pollution, and having high safety. It is compatible with all conventional fluorescence quantitative PCR instruments, easy to operate, simple and fast, saving costs and time for medical detection.

[0117] (3) The detection system of this application has strong inclusiveness. For the pathogenic bacteria of mucormycosis, common pathogenic bacteria such as Rhizopus, Rhizomucor, Mucor, Absidia, Cunninghamella bertholletiae, etc. can be detected, effectively providing test results and medication guidance for mucormycosis patients.

[0118] (4) The detection sensitivity of this application is strong, and the sensitivity of each target can reach 10 copies per reaction, which can effectively improve the problems of misdiagnosis, missed diagnosis and delayed treatment in medical diagnosis.

[0119] The implementation scheme of this application will be described in detail below in combination with embodiments. It should be understood that these embodiments are only used to illustrate this application and not to limit the scope of this application. For the experimental methods without specific conditions in the following embodiments, the guidance given in this application shall be preferentially referred to, and it can also be carried out according to the experimental manuals or conventional conditions in this field, or according to the conditions recommended by the manufacturer, or referring to the experimental methods known in this field.

[0120] In the following specific embodiments, for the measurement parameters of raw material components, if there is no special description, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0121] It should be understood that in various embodiments of the present application, the sequence numbers of the above processes do not indicate the order of execution. The order of execution of each process should be determined according to its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

[0122] Example 1

[0123] 1. The primer-probe combination for detecting 14 pathogenic fungi in the present application includes THO detection probes, PCO detection probes, and primer pairs, and the sequences are shown in Table 1.

[0124] The nucleic acid combination product for 14 pathogenic fungi further includes the following reagents:

[0125] ① Negative control: DNase / RNase-free water;

[0126] ② Positive control: Plasmid containing Candida albicans, Cryptococcus, Aspergillus fumigatus, and internal standard fragments;

[0127] ③ Enzyme mixture (PhyNexus, MDAD025-1).

[0128] 2. The reagent parameters are shown in Table 2:

[0129] Table 2

[0130] Final concentration of primer Final concentration of probe Concentration of enzyme mixture 200 nM 100 nM 1×

[0131] 3. The reagent formula is shown in Table 3:

[0132] Table 3

[0133] Main components Added amount (μL) / test Enzyme mixture 4 Working solution of primer and probe 2 Enzyme-free water 9 Total 15

[0134] (1) Take out the kit, fully thaw it at room temperature, mix all components, then centrifuge quickly, and place it on an ice box for standby.

[0135] (2) Calculate the number of tests required for the current time (n = number of samples + positive control + negative control + 1), and prepare the reaction solution according to the formula in Table 3.

[0136] (3) Mix the prepared PCR reaction solution, centrifuge quickly, dispense 15 μL per well into the corresponding PCR tubes, and transfer them to the sample processing area for use.

[0137] 4. Sample processing (sample processing area)

[0138] In this example, the sample source for detection is sputum. The sputum liquefaction reagent and nucleic acid extraction and purification kit produced by Guangzhou Baochuang Biotechnology Co., Ltd. are used for liquefying sputum samples and extracting sample nucleic acids.

[0139] (1) Liquefy the sputum. According to the volume and consistency of the sputum, add twice the amount of sputum liquefaction reagent to make the sputum finally in a non-stringy state, indicating complete liquefaction.

[0140] (2) Resuspend and incubate. Take 200 μL of completely liquefied sputum, add 20 μL of resuspension solution, vortex for 25 s, and incubate at 95 °C for 20 min.

[0141] (3) Sample addition. Take out the pre-packaged kit, invert and mix several times to resuspend the magnetic beads, then gently flick or briefly centrifuge the microplate to concentrate the reagent and magnetic beads at the bottom of the microplate. Carefully tear off the sealing film of the microplate, then add 20 μL of proteinase K solution to the 1st or 7th of the pre-packaged reagent, and then take 200 μL of the sample and add it thereto.

[0142] (4) On-machine extraction. Place the deep well plate in the nucleic acid extractor and run the preset "Germ-Fast" process according to the extract reagent instruction manual for automated extraction.

[0143] (5) Nucleic acid collection. After the automated extraction is completed, the 6th or 12th column is the nucleic acid after extraction. Transfer the liquid therein to a centrifuge tube for subsequent detection or storage at -20 °C or below.

[0144] 5. Sample addition (sample processing area)

[0145] Add 5 μL of negative control, sample extract, and positive control to the reaction solution in sequence, tighten the reaction tube, make good marks, centrifuge for 30 s, and transfer to the PCR amplification area.

[0146] 6. PCR amplification (amplification area)

[0147] Set the PCR reaction program as shown in Table 4 below, and set the amplification volume to 20 μL.

[0148] Table 4

[0149]

[0150] 7. Result determination criteria:

[0151] (1) Determination of baseline: The baseline automatically adjusted by the instrument can be used, or the user can also adjust the baseline according to the actual amplification background signal;

[0152] (2) Determination of threshold: The threshold is generally set at the inflection point of the exponential amplification period of the sample and is higher than the baseline background. It can be adjusted according to the actual amplification situation, and the baselines and thresholds of different channels should be determined separately;

[0153] (3) Use the control products in the kit for quality control

[0154] Positive control: FAM, VIC, ROX, and CY5 all have amplification curves and Ct ≤ 35. At the same time, the melting curve typing corresponding to FAM, VIC, and ROX is Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus.

[0155] Negative control: There is no amplification curve in each channel or Ct value > 40.

[0156] The test results of the sample can be interpreted only after the negative control and positive control are judged to be qualified.

[0157] (4) Interpretation method:

[0158] First, analyze whether there is amplification of the internal reference in the CY5 channel. If there is no amplification in the CY5 channel or Ct value > 35, it means that the concentration of the test sample is too low or there are interfering substances inhibiting the reaction, and the experiment needs to be prepared again. If there is amplification in CY5 and Ct value ≤ 35, it means that the current result is valid and the subsequent analysis can be continued.

[0159] A) If there is amplification in the FAM channel and Ct ≤ 40, it means that the FAM target is positive, and the target type needs to be judged through the characteristic melting curve; if there is a characteristic melting curve at Tm (28 - 31°C), it means that Candida tropicalis is positive; if there is a characteristic melting curve at Tm (34 - 37°C), it means that Candida albicans is positive; if there is a characteristic melting curve at Tm (41 - 44°C), it means that Candida glabrata is positive; if there is a characteristic melting curve at Tm (47 - 50°C), it means that Candida krusei is positive; if there is a characteristic melting curve at Tm (54 - 57°C), it means that Candida lusitaniae is positive;

[0160] B) If there is amplification in the VIC channel and Ct ≤ 40, it means that the VIC target is positive, and the target type needs to be judged through the characteristic melting curve; if there is a characteristic melting curve at Tm (31 - 34°C), it means that Candida auris is positive; if there is a characteristic melting curve at Tm (41 - 44°C), it means that Cryptococcus neoformans is positive; if there is a characteristic melting curve at Tm (49 - 52°C), it means that Pneumocystis jirovecii is positive; if there is a characteristic melting curve at Tm (55.5 - 58.5°C), it means that the Candida parapsilosis complex is positive;

[0161] C) If there is amplification in the ROX channel and Ct ≤ 40, it means that the ROX target is positive, and the target type needs to be judged through the characteristic melting curve; if there is a characteristic melting curve at Tm (30 - 33°C), it means that the Aspergillus flavus complex is positive; if there is a characteristic melting curve at Tm (37 - 40°C), it means that the causative agent of mucormycosis is positive; if there is a characteristic melting curve at Tm (42 - 45°C), it means that the Aspergillus terreus complex is positive; if there is a characteristic melting curve at Tm (48.5 - 51.5°C), it means that the Aspergillus fumigatus complex is positive; if there is a characteristic melting curve at Tm (55.5 - 58.5°C), it means that the Aspergillus niger complex is positive.

[0162] Example 2 Sensitivity Verification

[0163] In this example, plasmids of Candida tropicalis, Candida albicans, Candida glabrata, Candida krusei, Candida lusitaniae, Candida auris, Cryptococcus neoformans, Pneumocystis jirovecii, Candida parapsilosis, pathogenic fungi of mucormycosis, Aspergillus flavus, Aspergillus terreus, Aspergillus fumigatus, Aspergillus niger, etc. with known values were used to test the sensitivity of the primer-probe combination reagent of this application.

[0164] Specifically, 14 nucleic acid samples were serially diluted to 2000 copies / mL for detection, that is, 10 copies per reaction, and at the same time, sterilized purified water was used as a negative control.

[0165] The results are shown in Figures 1 to 14 . Figure 1 is the amplification curve and melting curve characteristic map of Candida tropicalis when the template detection quantity in the reaction system is 10 copies; Figure 2 is the amplification curve and melting curve characteristic map of Candida albicans when the template detection quantity in the reaction system is 10 copies; Figure 3 is the amplification curve and melting curve characteristic map of Candida glabrata when the template detection quantity in the reaction system is 10 copies; Figure 4 is the amplification curve and melting curve characteristic map of Candida krusei when the template detection quantity in the reaction system is 10 copies; Figure 5 is the amplification curve and melting curve characteristic map of Candida lusitaniae when the template detection quantity in the reaction system is 10 copies; Figure 6 is the amplification curve and melting curve characteristic map of Candida auris when the template detection quantity in the reaction system is 10 copies; Figure 7 is the amplification curve and melting curve characteristic map of Cryptococcus neoformans when the template detection quantity in the reaction system is 10 copies; Figure 8 is the amplification curve and melting curve characteristic map of Pneumocystis jirovecii when the template detection quantity in the reaction system is 10 copies; Figure 9 is the amplification curve and melting curve characteristic map of Candida parapsilosis when the template detection quantity in the reaction system is 10 copies; Figure 10 is the amplification curve and melting curve characteristic map of Aspergillus flavus complex when the template detection quantity in the reaction system is 10 copies; Figure 11 is the amplification curve and melting curve characteristic map of pathogenic fungi of mucormycosis when the template detection quantity in the reaction system is 10 copies; Figure 12 is the amplification curve and melting curve characteristic map of Aspergillus terreus complex when the template detection quantity in the reaction system is 10 copies; Figure 13Amplification curve and melting curve characteristic maps of Aspergillus fumigatus complex when the number of template detections in the reaction system is 10 copies; Figure 14 Amplification curve and melting curve characteristic maps of Aspergillus niger complex when the number of template detections in the reaction system is 10 copies. As Figures 11 to 14 shown, the minimum detection limit of each target can reach 10 copies reaction. It shows that the sensitivity of this reagent is good.

[0166] Example 3 Specificity verification

[0167] Cross-reaction detection was carried out on human-derived fungi that are close to and susceptible to the detection targets of the above kit outside the detection range of the above kit. It includes 10 negative control products of Candida guilliermondii, Aspergillus ustus, Aspergillus nidulans, Aspergillus sydowii, Aspergillus versicolor, Fusarium spp., Talaromyces spp., Penicillium brevicompactum, Penicillium oxalicum, Penicillium chrysogenum.

[0168] The results are as Figure 15 shown. The kit prepared in this application has no amplification curve for the above 10 samples outside the detection range of the kit, and they are all negative, indicating that the reagent of this application has good specificity and no cross-reaction.

[0169] Example 4 Inclusivity verification

[0170] The common pathogenic bacteria of Rhizopus, Rhizomucor, Mucor, Absidia, and Cunninghamella bertholletiae, which are common in the pathogenic bacteria of mucormycosis, the detection targets of the above kit, were detected. It includes 9 quality control products of pathogenic bacteria of mucormycosis, namely Mucor racemosus, Mucor circinelloides, Rhizomucor miehei, Rhizomucor pusillus, Rhizopus oryzae, Rhizopus arrhizus, Absidia coerulea, Absidia corymbifera, and Cunninghamella elegans.

[0171] The results are as Figure 16 shown. The kit prepared in this application has amplification curves for the above 9 quality control products and melting curve characteristic maps of pathogenic bacteria of mucormycosis, indicating that the reagent has strong inclusivity for pathogenic bacteria of mucormycosis.

[0172] The above-described embodiments merely represent several implementation manners of the present application, facilitating a specific and detailed understanding of the technical solutions of the present application. However, it should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can be made, and these all fall within the protection scope of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning, or limited experiments based on the technical solutions provided by the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the content of the appended claims, and the specification can be used to explain the content of the claims.

Claims

1. A nucleic acid combination product, characterized in that: The nucleic acid combination product includes multiple groups of the following probe primer combinations: (1) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 2, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 3, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 4; (2) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 2, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 5, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 6; (3) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 2, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 7, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 8; (4) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 2, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 9, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 10; (5) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 2, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 11, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 12; (6) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 2, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 13, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 14; (7) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 1 to SEQ ID NO: 2, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 15, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 16; (8) a primer pair having a nucleotide sequence as shown in SEQ ID NO: 17 to SEQ ID NO: 18, a THO probe having a nucleotide sequence as shown in SEQ ID NO: 19, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO: 20; (9) a primer pair having a nucleotide sequence as shown in SEQ ID NO:21 to SEQ ID NO:22, a THO probe having a nucleotide sequence as shown in SEQ ID NO:23, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO:24; (10) a primer pair having a nucleotide sequence as shown in SEQ ID NO:25 to SEQ ID NO:26, a THO probe having a nucleotide sequence as shown in SEQ ID NO:27, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO:28; (11) a primer pair having a nucleotide sequence as shown in SEQ ID NO:29 to SEQ ID NO:30, a THO probe having a nucleotide sequence as shown in SEQ ID NO:31, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO:32; (12) a primer pair having a nucleotide sequence as shown in SEQ ID NO:29 to SEQ ID NO:30, a THO probe having a nucleotide sequence as shown in SEQ ID NO:33, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO:34; (13) a primer pair having a nucleotide sequence as shown in SEQ ID NO:29 to SEQ ID NO:30, a THO probe having a nucleotide sequence as shown in SEQ ID NO:35, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO:36; (14) A primer pair having a nucleotide sequence as shown in SEQ ID NO:29 to SEQ ID NO:30, a THO probe having a nucleotide sequence as shown in SEQ ID NO:37, and a PCO probe having a nucleotide sequence as shown in SEQ ID NO:

38.

2. The nucleic acid combination product according to claim 1, characterized in that: The nucleic acid combination product includes primers and probes with nucleotide sequences as shown in SEQ ID NO: 1 to SEQ ID NO:

38.

3. The nucleic acid combination product according to claim 1, characterized in that: The 3' end of the PCO detection probe is labeled with a phosphate group; the two ends of the THO detection probe are respectively labeled with a fluorescent reporter group and a fluorescent quencher group; Optionally, the 5' end of the THO detection probe is labeled with the fluorescent reporter group, and the 3' end is labeled with the fluorescent quencher group; Optionally, the fluorescent reporter group includes one or more of FAM, VIC, ROX and CY5; Optionally, the fluorescence quenching group includes one or more of BHQ1 and BHQ2.

4. The nucleic acid combination product according to claim 1, characterized in that: The nucleic acid combination product also includes an internal reference gene detection primer pair and / or an internal reference gene detection probe; Optionally, the internal reference gene detection primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO.39 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.40; Optionally, the nucleotide sequence of the internal reference gene detection probe is shown in SEQ ID NO.

41.

5. A kit for detecting pathogenic fungi, characterized in that: The kit comprises: the nucleic acid combination product according to any one of claims 1 to 4.

6. The kit according to claim 5, characterized in that The kit further comprises: one or more of a nucleic acid releasing reagent, a nucleic acid extracting reagent and a nucleic acid amplifying reagent; Optionally, the nucleic acid amplification reagent includes DNA polymerase, dNTPs, UNG enzyme, PCR buffer and Mg 2+ One or more of .

7. The kit according to claim 5 or 6, characterized in that The kit also includes one or more of a positive quality control product and a negative quality control product.

8. The kit according to claim 7, characterized in that The positive quality control product includes one or more of Candida albicans quality control product, Cryptococcus quality control product and Aspergillus fumigatus quality control product; Or / and, the negative control product includes enzyme-free water.

9. A method for detecting pathogenic fungi, characterized in that: include: Providing a nucleic acid sample to be tested, using the nucleic acid combination product according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 8 to perform a PCR amplification reaction on the nucleic acid sample to be tested, analyzing the obtained amplification product using a melting curve method, and determining whether the nucleic acid sample to be tested contains pathogenic fungi based on the obtained analysis results; Optionally, the PCR amplification reaction includes an MPA multiplex fluorescence PCR amplification reaction.

10. The method for detecting pathogenic fungi according to claim 9, characterized in that: The source of the nucleic acid sample to be tested includes a sputum sample.

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