Primer probe combination, method and kit for detecting cryptococcal subtypes

Abstract

The present application relates to a primer-probe combination, method and kit for detecting cryptococcal subtypes. The primer includes a first primer, the first primer includes a first forward primer and a first reverse primer, the nucleotide sequence of the first forward primer is shown in Seq ID No. 1, the nucleotide sequence of the first reverse primer is shown in Seq ID No. 2, and the probe includes a first probe, the nucleotide sequence of the first probe is shown in Seq ID No. 3. The primer-probe combination provided in the present application can accurately and quickly detect cryptococcal subtypes, help improve the rapid diagnosis and treatment of cryptococcal subtypes that are susceptible to disease, and buy precious time for the treatment of the disease.

Description

Technical Field

[0001] The invention belongs to the technical field of fungal pathogen detection, and in particular relates to a primer-probe combination, a method and a kit for detecting cryptococcal subtypes. Background Art

[0002] In recent years, cryptococcal infections have been on the rise both at home and abroad. Cryptococcus is a type of fungus that can cause cryptococcosis. The genus Cryptococcus includes 17 neutral and 8 variants. There are significant differences in the morbidity and infected populations of different species of Cryptococcus. With the improvement of genetic testing methods, Cryptococcus also has different molecular typing, such as VGI cryptococcus, VGII cryptococcus, etc. There are also significant differences in the prevalence, clinical manifestations, diagnosis and treatment of different types of cryptococci. Therefore, it is crucial for clinical diagnosis to identify which type of cryptococcus causes the infection.

[0003] Although existing cryptococcal detection methods can detect cryptococci, most of them can only detect cryptococci and cannot further determine the molecular typing of cryptococci. Therefore, how to accurately detect the subtypes of cryptococci is a technical problem that needs to be solved urgently. Summary of the invention

[0004] The present invention provides a primer-probe combination, method, kit for detecting cryptococcal subtypes and application of the primer-probe combination in detecting cryptococcal subtypes. By designing corresponding primer-probe combinations for specific gene fragments of multiple cryptococcal subtypes, cryptococcal subtypes can be accurately detected, thereby facilitating early clinical diagnosis of cryptococcal disease.

[0005] In a first aspect, the present application provides a primer-probe combination for detecting cryptococcal subtypes, the primers comprising a first primer, the first primer comprising a first forward primer and a first reverse primer, the nucleotide sequence of the first forward primer being as shown in Seq ID No.1, and the nucleotide sequence of the first reverse primer being as shown in Seq ID No.2; the probe comprising a first probe, the nucleotide sequence of the first probe being as shown in Seq ID No.3.

[0006] In some embodiments, the first primer is used to identify the nucleotide sequence shown in Seq ID No. 4 in VGIb type Cryptococcus.

[0007] In some embodiments, the 5' end of the first probe is connected to a first fluorescent reporter group, and the 3' end of the first probe is connected to a first fluorescent quencher group.

[0008] In some embodiments, the primer includes a second primer, the second primer includes a second forward primer and a second reverse primer, the nucleotide sequence of the second forward primer is as shown in Seq ID No.5, and the nucleotide sequence of the second reverse primer is as shown in Seq ID No.6; the probe includes a second probe, and the nucleotide sequence of the second probe is as shown in Seq ID No.7.

[0009] In some embodiments, the second primer is used to recognize the nucleotide sequence shown in Seq ID No. 8 in VGIa type Cryptococcus.

[0010] In some embodiments, the 5' end of the second probe is connected to a second fluorescent reporter group, and the 3' end of the second probe is connected to a second fluorescent quencher group.

[0011] In a second aspect, a method for detecting cryptococcal subtypes is provided, the method comprising: obtaining a template DNA of a sample to be tested; amplifying the template DNA by polymerase chain reaction (PCR) to obtain an amplification result, wherein the PCR reactants include the primer-probe combination in any embodiment of the first aspect; and determining the subtype of cryptococcus in the sample to be tested based on the amplification result.

[0012] In some embodiments, the cryptococcus includes at least one of VGIa type cryptococcus, VGIb type cryptococcus, and wild cryptococcus.

[0013] It should be understood that wild cryptococci refer to other types of cryptococci except VGIa type cryptococci and VGIb type cryptococci.

[0014] In some embodiments, determining the subtype of the Cryptococcus in the sample to be tested based on the amplification result includes: when the first fluorescent reporter group has a logarithmic increase in fluorescence in the amplification result and the cycle threshold Ct is less than or equal to 30.0, determining that the Cryptococcus in the sample to be tested is a VGIb type Cryptococcus; when the second fluorescent reporter group has a logarithmic increase in fluorescence in the amplification result and the cycle threshold Ct is less than or equal to 30.0, determining that the Cryptococcus in the sample to be tested is a VGIa type Cryptococcus.

[0015] In a third aspect, a kit for detecting cryptococcal subtypes is provided, wherein the kit comprises the primer-probe combination in any embodiment of the first aspect.

[0016] In some embodiments, the kit further comprises: a buffer, a template DNA, a DNA polymerase and / or deoxynucleotide triphosphate dNTPs.

[0017] In a fourth aspect, there is provided a use of a primer-probe combination as in any embodiment of the first aspect in detecting Cryptococcus. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic flow chart of a method for detecting a cryptococcal subtype in an embodiment of the present application.

[0019] Figure 2 This is a specific amplification curve of VGIb type Cryptococcus in the example of the present application.

[0020] Figure 3 This is a specific amplification curve of VGIa type Cryptococcus in the example of the present application. DETAILED DESCRIPTION

[0021] The technical solution of the present application is further introduced below in conjunction with specific embodiments.

[0022] "Scope" disclosed in the present application is limited in the form of lower limit and upper limit, and a given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a special range. The scope limited in this way can be including end values ​​or not including end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a scope. For example, if the scope of 60-120 and 80-110 is listed for a specific parameter, it is understood that the scope of 60-110 and 80-120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following scope can be all expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In the present application, unless otherwise specified, the numerical range "ab" represents the abbreviation of any real number combination between a and b, wherein a and b are real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0023] In the description of the present application, the terms “first”, “second”, “third”, etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance.

[0024] If there is no special explanation, the "include" and "comprising" mentioned in this application are open-ended or closed-ended. For example, the "include" and "comprising" may mean that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0025] If not specifically stated, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0026] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0027] Cryptococcus is a type of fungus widely found in nature, belonging to the subphylum Ascomycota, class Bacillus, and family Cryptococcaceae. The genus Cryptococcus includes 17 species and 8 variants, among which the main pathogenic one is Cryptococcus neoformans, hereinafter referred to as Cryptococcus. There are significant differences in the morbidity and infected populations of different types of Cryptococcus. For example, Cryptococcus neoformans mainly infects immunosuppressed people, while Cryptococcus gattii mainly infects immune-normal people.

[0028] With the improvement of genetic detection methods, Cryptococcus is divided into 8 types in the primary typing, including: VNI ​​type Cryptococcus, VNII type Cryptococcus, VNIII type Cryptococcus, VNIV type Cryptococcus, VGI type Cryptococcus, VGII type Cryptococcus, VGIII type Cryptococcus and VGIV type Cryptococcus. In addition, the inventors have found that under the above 8 typings, Cryptococcus can be further subdivided. For example, VGI type Cryptococcus can also be divided into multiple subtypes such as VGIa type Cryptococcus, VGIb type Cryptococcus, and VGIc type Cryptococcus. Clinical data show that the mortality rate of VGIb type Cryptococcus is significantly higher than that of other strains, which will lead to more serious clinical consequences compared with other strains. Therefore, how to detect and identify the subtypes of Cryptococcus is of great significance for clinical diagnosis.

[0029] In view of this, the present application provides a primer-probe combination for detecting cryptococcal subtypes. The polymerase chain reaction (PCR) based on the primer-probe combination can accurately and quickly detect cryptococci and identify cryptococcal subtypes.

[0030] First, the present application embodiment provides a primer-probe combination for detecting Cryptococcal subtypes.

[0031] The primers include a first primer, and the first primer includes a first forward primer and a first reverse primer. The nucleotide sequence of the first forward primer is shown in Seq ID No.1, and the nucleotide sequence of the first reverse primer is shown in Seq ID No.2.

[0032] The probe comprises a first probe, and the nucleotide sequence of the first probe is shown as Seq ID No.3.

[0033] Specifically, the inventors used bioinformatics methods to compare the entire genomes of various types of cryptococci, not limited to a single coding gene, but to find differential sites at the entire genome level, thereby designing a first primer with a nucleotide sequence as shown in Seq ID No. 1 and Seq ID No. 2 and a first probe with a nucleotide sequence as shown in Seq ID No. 3. The first primer and the first probe can accurately detect and identify VGIb type cryptococci, thereby helping to diagnose diseases caused by VGIb type cryptococci at an early stage.

[0034] It should be understood that the cryptococcus described in the present application includes VGIa type cryptococcus, VGIb type cryptococcus and wild-type cryptococcus, wherein wild-type cryptococcus refers to other types of cryptococcus except VGIa type cryptococcus and VGIb type cryptococcus.

[0035] The nucleotide sequence of the first forward primer is specifically:

[0036] GCTTAAGGGATTTTCTTGG.

[0037] The nucleotide sequence of the first reverse primer is specifically:

[0038] TCAGATGTGTGTCGGCCAGA.

[0039] The first primer is used to recognize the nucleotide sequence in VGIb cryptococcus as shown in Seq ID No. 4. In other words, the first primer designed for VGIb cryptococcus can specifically recognize the nucleotide sequence shown in Seq ID No. 4, which only exists in VGIb cryptococcus.

[0040] Optionally, the 5' end of the first probe is connected to a first fluorescent reporter group, and the 3' end of the first probe is connected to a first fluorescent quencher group.

[0041] The first fluorescent reporter group includes but is not limited to 6-carboxyfluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein (JOE), hexachloro-6-methylfluorescein (HEX), anthocyanin fluorescent dye CY3, 6-carboxy-tetramethylrhodamine (TAMRA). The first fluorescent quencher group includes but is not limited to 6-carboxy-tetramethylrhodamine (TAMRA), 4-(4-oxanthylaminophenylazo)benzoic acid (DABCYL) or black hole quenchers BHQ1, BHQ2, BHQ3.

[0042] For example, the 5' end of the first probe is connected to a FAM group, and the 3' end of the first probe is connected to a BHQ1 group. That is, the first probe is:

[0043] FAM-ATATGGCTGGAAACTGAACA-BHQ1.

[0044] Optionally, the primer includes a second primer, the second primer includes a second forward primer and a second reverse primer. The nucleotide sequence of the second forward primer is shown in Seq ID No.5, and the nucleotide sequence of the second reverse primer is shown in Seq ID No.6. The probe includes a second probe, and the nucleotide sequence of the second probe is shown in Seq ID No.7.

[0045] The second primer is used to recognize the nucleotide sequence shown in Seq ID No. 8 in VGIa type Cryptococcus.

[0046] Specifically, the primer-probe combination for detecting cryptococcal subtypes may also include a second primer and a second probe. The second primer and the second probe are designed for VGIa cryptococcus, and can specifically identify the nucleotide sequence of VGIa cryptococcus as shown in Seq ID No. 8, thereby accurately detecting VGIa cryptococcus. In other words, the second primer and the probe can not only accurately detect cryptococcus, but also determine that the detected cryptococcus is VGIa cryptococcus.

[0047] Wherein, the nucleotide sequence of the second forward primer is specifically:

[0048] GTTGCCCCAAAAATTGTATTATAC.

[0049] The nucleotide sequence of the second reverse primer is specifically:

[0050] AGTGAACTGAGACATGTTTCTT.

[0051] Optionally, the 5' end of the second probe is connected to a second fluorescent reporter group, and the 3' end of the second probe is connected to a second fluorescent quencher group.

[0052] The second fluorescent reporter group includes, but is not limited to, 6-carboxyfluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein (JOE), hexachloro-6-methylfluorescein (HEX), anthocyanin fluorescent dye CY3, 6-carboxy-tetramethylrhodamine (TAMRA). The second fluorescent quencher group includes, but is not limited to, 6-carboxy-tetramethylrhodamine (TAMRA), 4-(4-oxanylaminophenylazo)benzoic acid (DABCYL) or black hole quenchers BHQ1, BHQ2, BHQ3.

[0053] For example, the 5' end of the second probe is connected to a FAM group, and the 3' end of the second probe is connected to a BHQ1 group. That is, the second probe is:

[0054] FAM-ACCAAATCCTCCGCTTAACGTGATTTTTAACGT-BHQ1.

[0055] It should be understood that the first fluorescent reporter group and the second fluorescent reporter group may be the same or different. The first fluorescent quencher group and the second fluorescent quencher group may be the same or different.

[0056] The primer-probe combination provided in the embodiments of the present application may include only the first primer and the first probe, or only the second primer and the second probe, which are respectively used for efficient and accurate detection of VGIb type Cryptococcus or VGIa type Cryptococcus; it may also include the first primer, the second primer, the first probe and the second probe at the same time, which are used for simultaneous detection of VGIb type Cryptococcus and VGIa type Cryptococcus.

[0057] The primer-probe combination has the obvious advantages of high sensitivity and strong specificity in detecting and identifying cryptococcal subtypes. Furthermore, the primer-probe combination can also be used to identify the type of cryptococcus while detecting cryptococcus, thereby facilitating rapid diagnosis of cryptococcus in the early clinical stage.

[0058] The embodiment of the present application also provides a method 100 for detecting Cryptococcus.

[0059] Figure 1 1 is a schematic flow chart of a method 100 for detecting cryptococci according to an embodiment of the present application. Figure 1 As shown, the method 100 includes:

[0060] S101, obtaining template DNA of the sample to be tested.

[0061] S102, amplifying the template DNA of the sample to be tested by polymerase chain reaction (PCR) to obtain an amplification result, wherein the PCR reactants include the primer-probe combination in any embodiment of the present application.

[0062] S103, determining the subtype of Cryptococcus in the sample to be tested according to the amplification result.

[0063] Specifically, the primer-probe combination provided in the present application can effectively detect Cryptococcus in the sample to be tested and identify the Cryptococcus subtype in the sample to be tested through fluorescent quantitative PCR.

[0064] Optionally, the cryptococcus in method 100 includes at least one of VGIa cryptococcus, VGIb cryptococcus and wild-type cryptococcus, wherein the wild-type cryptococcus refers to other types of cryptococcus except VGIa cryptococcus and VGIb cryptococcus.

[0065] Optionally, the sample to be tested includes blood, body fluid, and / or lung tissue.

[0066] Optionally, in S102, the PCR reactants include a first primer and a first probe. In S103, judging the subtype of Cryptococcus in the sample to be tested according to the amplification result includes:

[0067] In the amplification result, when the first fluorescent reporter group has a fluorescence logarithmic increase and the cycle threshold Ct is less than or equal to 30.0, it is determined that the cryptococcus in the sample to be tested is a VGIb type cryptococcus.

[0068] In the amplification result, when the first fluorescent reporter group has no logarithmic fluorescence increase or the first fluorescent reporter group has a logarithmic fluorescence increase but the cycle threshold Ct is greater than 30.0, it is determined that the cryptococcus in the sample to be tested is not a VGIb type cryptococcus.

[0069] Optionally, in S102, the PCR reactants include a second primer and a second probe. In S103, judging the subtype of Cryptococcus in the sample to be tested according to the amplification result includes:

[0070] In the amplification result, when the second fluorescent reporter group has a fluorescence logarithmic increase and the cycle threshold Ct is less than or equal to 30.0, it is determined that the cryptococcus in the sample to be tested is a VGIa type cryptococcus.

[0071] In the amplification result, when the second fluorescent reporter group has no logarithmic fluorescence increase or the second fluorescent reporter group has a logarithmic fluorescence increase but the cycle threshold Ct is greater than 30.0, it is determined that the cryptococcus in the sample to be tested is not a VGIa type cryptococcus.

[0072] In practical applications, the sample to be tested is usually an unknown sample. The first primer and the first probe can be used to detect whether the cryptococcus in the sample to be tested is a VGIb type cryptococcus with a higher pathogenicity, and then the second primer and the second probe can be used to detect whether the cryptococcus in the sample to be tested is a VGIa type cryptococcus. The sample to be tested can also be tested by the above-mentioned multiple primers and multiple probes at the same time, and the subtype of the cryptococcus in the sample to be tested can be determined by combining and analyzing the detection results of each primer probe.

[0073] Optionally, in S102, the PCR reactants include a first primer, a second primer, a first probe, and a second probe. In S103, judging the subtype of Cryptococcus in the sample to be tested according to the amplification result includes:

[0074] When the first fluorescent reporter group has a logarithmic fluorescence increase in the amplification result and the cycle threshold Ct is less than or equal to 30.0, it is determined that the cryptococcus in the sample to be tested is a VGIb type cryptococcus.

[0075] When the second fluorescent reporter group has a logarithmic fluorescence increase in the amplification result and the cycle threshold Ct is less than or equal to 30.0, it is determined that the cryptococcus in the sample to be tested is a VGIa type cryptococcus.

[0076] Specifically, if the first fluorescent reporter group has a fluorescence logarithmic increase and the cycle threshold Ct is less than or equal to 30.0, and the second fluorescent reporter group has no fluorescence logarithmic increase or has a fluorescence logarithmic increase but the cycle threshold Ct is greater than 30.0, then it is judged that VGIb type cryptococci exist in the cryptococci in the sample to be tested and VGIa type cryptococci do not exist; if the second fluorescent reporter group has a fluorescence logarithmic increase and the cycle threshold Ct is less than or equal to 30.0, and the first fluorescent reporter group has no fluorescence logarithmic increase or has a fluorescence logarithmic increase but the cycle threshold Ct is greater than 30.0, then VGIa type cryptococci exist in the cryptococci in the sample to be tested and VGIb type cryptococci do not exist; if both the first fluorescent reporter group and the second fluorescent reporter group have fluorescence logarithmic increase and the cycle threshold Ct is less than or equal to 30.0, then it is judged that VGIa type cryptococci and VGIb type cryptococci exist in the cryptococci in the sample to be tested. In other cases, it is judged that there is no cryptococcus in the sample to be tested or the cryptococcus is a wild-type cryptococcus.

[0077] It should be understood that when the first fluorescent reporter group and the second fluorescent reporter group are the same group, fluorescent quantitative PCR can determine whether the first fluorescent reporter group and the second fluorescent reporter group have corresponding logarithmic growth by the specific amount of fluorescence logarithmic growth.

[0078] The embodiments of the present application also provide a kit for detecting Cryptococcal subtypes, wherein the kit comprises the primer-probe combination in any embodiment of the present application.

[0079] Optionally, the kit further comprises: a buffer, a template DNA, a DNA polymerase and / or deoxynucleotide triphosphate dNTP.

[0080] Optionally, the kit further comprises MgCl2.

[0081] The kit provided in the examples of the present application can accurately and quickly detect cryptococci. It is simple to operate, has a short detection time, and is highly adaptable. It can also identify the subtypes of pathogenic bacteria, thereby making a rapid and accurate clinical diagnosis and saving precious time for disease treatment.

[0082] In addition, an embodiment of the present application also provides an application of a primer-probe combination as described in any of the above embodiments in detecting Cryptococcal subtypes.

[0083] Next, the specific steps of the detection method 100 provided in the present application are further introduced. It should be understood that the following examples are only for illustrating the implementation of the identification method of the present application, wherein the specific reagent dosage or instrument usage can be adjusted as needed, and does not constitute a limitation on the examples of the present application.

[0084] Example 1: Extraction of template DNA

[0085] 1. Test materials: Samples to be tested

[0086] The samples to be tested described in the present application include but are not limited to blood, body fluids or lung tissues.

[0087] 2. Main reagents and instruments

[0088] DNA was extracted using the yeast gene extraction kit from Tiangen Company; the synthesis of primers and probes was completed by Bioengineering Co., Ltd.; DNA polymerase was purchased from Takara Company; and the fluorescent quantitative PCR instrument was purchased from Roche Company.

[0089] 3. Experimental Methods

[0090] DNA was extracted according to the instructions of the DNA extraction kit, and the DNA content in the sample was determined using a spectrophotometer, and the OD260 / OD280 ratio was determined. The sample to be tested provided in this embodiment contains Cryptococcus neoformans, VGIa type Cryptococcus, and VGIb type Cryptococcus.

[0091] Example 2: PCR amplification reaction

[0092] 2.1 PCR detection of VGIb cryptococci

[0093] 1. Test Materials

[0094] Template DNA. The specific PCR reaction system is shown in Table 1.

[0095] 2. Main reagents and instruments

[0096] Table 1: Real-time fluorescence PCR reaction system 1 (25 μL)

[0097]

[0098] 3. Experimental Methods

[0099] In this example, the PCR amplification reaction was performed using the Prime STAR 2X Mix PCR amplification kit of TAKARA Company. The PCR amplification process can be found in the product instruction manual of the kit.

[0100] In this embodiment, the nucleotide sequence of the first forward primer in the reaction system 1 is shown in Seq ID No.1, the nucleotide sequence of the first reverse primer is shown in Seq ID No.2, and the nucleotide sequence of the first probe is shown in Seq ID No.3.

[0101] In this embodiment, the amplification conditions of the PCR reaction are: 95 o C pre-denaturation for 5 min; 95 o C 10s, 68 o C for 60 s and 45 cycles to obtain the amplification results.

[0102] 4. Analyze and judge the amplification results

[0103] Figure 2 This is the amplification curve of VGIb type Cryptococcus obtained through the above operation in the example of the present application. Figure 2 In the amplification curve, the ordinate is the relative fluorescence unit (RFU) and the abscissa is the number of amplification cycles.

[0104] like Figure 2 As shown, in the test results of the sample to be tested, only VGIb type Cryptococcus was specifically amplified, with a corresponding amplification curve, FAM fluorescence had a logarithmic increase in fluorescence, and Ct≤30.0. However, the VGIb type Cryptococcus and Cryptococcus neoformans in parallel experiments had no specific amplification curves. This shows that the first primer and the first probe have good specificity for VGIb type Cryptococcus and can accurately identify VGIb type Cryptococcus in the sample to be tested. Moreover, the three amplification curves of VGIb type Cryptococcus have almost the same logarithmic increase in fluorescence, indicating that the method of detecting VGIb type Cryptococcus by the first primer and the first probe has good repeatability. In addition, Figure 2 The template DNA concentration in the culture medium was 0.1 ng / μL, and there was still a good fluorescence logarithmic response after fluorescence PCR, indicating that the method of detecting VGIb type cryptococcus using the first primer and the first probe has excellent sensitivity.

[0105] Therefore, the primer-probe combination provided in the present application can accurately and efficiently detect VGIb type Cryptococcus.

[0106] 2.2 PCR detection of VGIb cryptococci

[0107] 1. Test materials: Template DNA. The specific PCR reaction system is shown in Table 2.

[0108] 2. Main reagents and instruments

[0109] Table 2: Real-time fluorescence PCR reaction system 2 (25 μL)

[0110]

[0111] 3. Experimental Methods

[0112] In this example, the PCR amplification reaction was performed using the Prime STAR 2X Mix PCR amplification kit of TAKARA Company. The PCR amplification process can be found in the product instruction manual of the kit.

[0113] In this embodiment, the nucleotide sequence of the second forward primer in reaction system 2 is shown in Seq ID No.5, the nucleotide sequence of the second reverse primer is shown in Seq ID No.6, and the nucleotide sequence of the second probe is shown in Seq ID No.7.

[0114] In this embodiment, the amplification conditions of the PCR reaction are: 95 o C pre-denaturation for 5 min; 95 o C 10s, 68 o C for 60 s and 45 cycles to obtain the amplification results.

[0115] 4. Analyze and judge the amplification results

[0116] Figure 3 This is the amplification curve of VGIa type Cryptococcus obtained through the above operation in the example of the present application. Figure 3 In the amplification curve, the ordinate is the relative fluorescence unit (RFU) and the abscissa is the number of amplification cycles.

[0117] like Figure 3 As shown, in the test results of the sample to be tested, VGIa type Cryptococcus was specifically amplified, with a corresponding amplification curve, FAM fluorescence had a logarithmic increase in fluorescence, and Ct≤30.0. However, the VGIb type Cryptococcus and Cryptococcus neoformans in parallel experiments had no specific amplification curves. This shows that the second primer and the second probe can specifically identify VGIa type Cryptococcus in the sample to be tested, with good specificity. And the three amplification curves of VGIa type Cryptococcus all have almost the same logarithmic increase in fluorescence, indicating that the method of detecting VGIa type Cryptococcus by the second primer and the second probe has good repeatability. In addition, Figure 3 The template DNA in the sample was 0.1 ng / μL, and there was still a good fluorescence logarithmic response after fluorescence PCR, indicating that the method of detecting VGIa type cryptococcus using the second primer and the second probe has excellent sensitivity.

[0118] Therefore, the primer-probe combination provided in the present application can accurately detect VGIa type Cryptococcus.

[0119] Through the above-mentioned detection of VGIa type cryptococcus, VGIb type cryptococcus and cryptococcus neoformans, it can be seen that the primer probe combination provided by the present application can specifically identify VGIa type cryptococcus and VGIb type cryptococcus, and the results of other strains in parallel experiments are all negative, without specific amplification curves, thus, the primer probe combination has good specificity and can accurately detect VGIa type cryptococcus and VGIb type cryptococcus. In addition, when the primer probe combination provided by the present application is used for cryptococcal detection, it is not only possible to detect whether there is cryptococcus, but also possible to directly determine the subtype of cryptococcus, which is conducive to clinically helping to carry out early intervention and treatment of cryptococcal subspecies with higher morbidity or mortality. The detection kit provided by the present application detects cryptococcal subtypes, which is simple to operate and the results are reliable and accurate, which can help doctors to quickly diagnose diseases infected by cryptococcus, and is conducive to formulating suitable treatment methods as soon as possible to reduce drug abuse.

[0120] In the description of this specification, the description with reference to the terms "one embodiment", "an implementation", "some embodiments", "illustrative implementation", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the implementation or example are included in at least one implementation or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same implementation or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more implementations or examples in a suitable manner.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A primer-probe combination for detecting cryptococcal subtypes, characterized in that: The primers include a first primer, which includes a first forward primer and a first reverse primer, the nucleotide sequence of the first forward primer is shown in Seq ID No.1, and the nucleotide sequence of the first reverse primer is shown in Seq ID No.2; the probe includes a first probe, and the nucleotide sequence of the first probe is shown in Seq ID No.

3.

2. The primer-probe combination according to claim 1, characterized in that: The 5' end of the first probe is connected to a first fluorescent reporter group, and the 3' end of the first probe is connected to a first fluorescent quencher group.

3. The primer-probe combination according to claim 1 or 2, characterized in that: The primers include a second primer, the second primer includes a second forward primer and a second reverse primer, the nucleotide sequence of the second forward primer is as shown in Seq ID No.5, and the nucleotide sequence of the second reverse primer is as shown in Seq ID No.6; the probe includes a second probe, and the nucleotide sequence of the second probe is as shown in Seq ID No.

7.

4. The primer-probe combination according to claim 3, characterized in that: The 5' end of the second probe is connected to a second fluorescent reporter group, and the 3' end of the second probe is connected to a second fluorescent quencher group.

5. A kit for detecting cryptococcal subtypes, characterized in that: The kit comprises the primer-probe combination according to any one of claims 1 to 4.

6. The kit according to claim 5, characterized in that The kit further comprises: a buffer, a DNA polymerase and / or deoxynucleoside triphosphate dNTP.

Images