Primer and probe for detecting talaromyces marneffei, application and kit

Abstract

The invention relates to the technical field of biological detection, in particular to a primer and a probe for detecting talaromyces marneffei, application and a kit. The primer and the probe for detecting talaromyces marneffei comprise an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO. 2; and the nucleotide sequence of the probe is as shown in SEQ ID NO.3. The talaromyces marneffei can be rapidly and accurately detected by using the primer and the probe based on droplet digital PCR, the use amount of a sample to be detected is small, pathogenic culture is not needed, time consumption is short, cross reaction does not exist in detection of other pathogenic bacteria, specificity is good, and sensitivity is high. Therefore, the kit has important application value in early diagnosis and treatment of talaromyces marneffei.

Description

Technical Field

[0001] The present application belongs to the field of biological detection technology, and in particular relates to a primer and a probe, an application and a kit for detecting Talaromyces marneffei. Background Art

[0002] Talaromyces marneffei is an opportunistic pathogenic fungus mainly distributed in Southeast Asian countries and southern China. People with low immunity, especially AIDS patients, are susceptible. In recent years, with the increase in AIDS patients, the number of cases of Talaromyces marneffei infection has also increased year by year, and the epidemic area has continued to expand. Talaromyces marneffei is prone to cause disseminated infection, with severe illness and high mortality. Early diagnosis and timely treatment are the key to reducing mortality.

[0003] Currently, positive pathogen culture is the gold standard for diagnosing Talaromycosis marneffei, but it usually takes 3 to 14 days, and early diagnosis is difficult. Serological diagnostic methods such as galactomannan (GM) test and mannan protein (Mp1p) antigen detection are rapid and simple, but lack sensitivity and specificity, and are generally only used for auxiliary diagnosis. Summary of the invention

[0004] The purpose of the present application is to provide a primer and a probe, an application and a kit for detecting Talaromyces marneffei, aiming to solve the technical problem of how to better detect Talaromyces marneffei.

[0005] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0006] In a first aspect, the present application provides a primer and a probe for detecting Talaromyces marneffei, comprising:

[0007] Upstream primer, the nucleotide sequence is shown in SEQ ID NO.1;

[0008] Downstream primer, nucleotide sequence is shown in SEQ ID NO.2;

[0009] and a probe, wherein the nucleotide sequence is shown in SEQ ID NO.3.

[0010] In some embodiments, the final concentrations of the primers and probes for detecting Talaromyces marneffei are: 0.25-1.0 μM for the upstream primer, 0.25-1.0 μM for the downstream primer, and 0.10-0.40 μM for the probe.

[0011] In some embodiments, the final concentrations of the primers and probes for detecting Talaromyces marneffei are: 0.5 μM for the upstream primer, 0.5 μM for the downstream primer, and 0.25 μM for the probe.

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

[0013] In some embodiments, the fluorescent reporter group includes at least one of FAM, VIC, HEX, TET, JOE, CY3, and CY5;

[0014] And / or, the fluorescence quenching group includes at least one of TAMARA, MGB, BHQ-1, BHQ-2 and BHQ-3.

[0015] In a second aspect, the present application provides an application, namely, the application of the primers and probes provided in the first aspect of the present application in the preparation of a kit for detecting Talaromyces marneffei based on droplet digital PCR.

[0016] In a third aspect, the present application provides a kit for detecting Talaromyces marneffei, the kit comprising the primers and probes provided in the first aspect of the present application.

[0017] In some embodiments, the kit detects Talaromyces marneffei based on droplet digital PCR.

[0018] In some embodiments, the kit further comprises: droplet digital PCR system reagents.

[0019] In some embodiments, the kit further comprises a sample nucleic acid extraction reagent.

[0020] The primers and probes for detecting Talaromyces marneffei provided in the first aspect of the present application are designed based on the ITS (Internal Transcribed Spacer) sequence of Talaromyces marneffei, and are highly specific primers and probes that can amplify the target sequence based on droplet digital PCR (Droplet Digital Polymerase Chain Reaction). The primers and probes can be used to quickly and accurately detect Talaromyces marneffei, which not only requires a small amount of sample to be detected, does not require pathogenic culture, and is time-consuming, but also has no cross-reaction to other pathogens, has good specificity, and high sensitivity. Therefore, it has important application value for the early diagnosis and treatment of Talaromyces marneffei.

[0021] The application provided in the second aspect of the present application is based on the fact that the primers and probes provided in the first aspect of the present application can quickly, accurately and sensitively detect Talaromyces marneffei based on droplet digital PCR. Therefore, the primers and probes can be used to prepare a kit for detecting Talaromyces marneffei based on droplet digital PCR.

[0022] The kit for detecting Talaromyces marneffei provided in the third aspect of the present application contains primers and probes unique to the present application. Based on the characteristics of the primers and probes, the kit can quickly, accurately and sensitively detect the Talaromyces marneffei pathogen, providing the possibility for early diagnosis of Talaromyces marneffei and grassroots epidemiological surveys. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 is an amplification curve graph corresponding to the primers and comparative primers provided in the examples of the present application;

[0025] Figure 2 It is the melting peak graph corresponding to the primers and comparative primers provided in the examples of the present application;

[0026] Figure 3 This is a graph showing the results of detecting different pathogens based on droplet digital PCR using the primers and probes provided in the examples of the present application. DETAILED DESCRIPTION

[0027] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0028] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0029] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0030] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0031] The terms "first", "second", etc. are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" or "second" may explicitly or implicitly include one or more of the features.

[0032] Droplet digital PCR is a nucleic acid detection technology. Its principle is to evenly disperse the PCR reaction system into tens of thousands of nanoliter droplets, each of which contains one to several target genes, or no target gene. After the PCR amplification is completed, each droplet is marked as positive or negative according to the fluorescence signal, and the copy number of the target gene is calculated based on the Poisson distribution principle and the number and proportion of positive droplets. Compared with fluorescent quantitative PCR, droplet digital PCR has better sensitivity, specificity and sample tolerance, and can perform absolute quantification of target genes without relying on standard curves. In addition, droplet digital PCR is easy to operate and has a short detection cycle. In recent years, droplet digital PCR has been successfully applied to tumor gene detection, genetic disease diagnosis, pathogen diagnosis and other fields.

[0033] However, the probes and primers designed for the target sequence in droplet digital PCR become the key. At present, there are no droplet digital PCR detection primers and probes and amplification systems for Talaromyces marneffei. In view of this, the present application designs a primer probe and its application and kit for detecting Talaromyces marneffei based on droplet digital PCR, which can quickly diagnose Talaromyces marneffei infection with high sensitivity and strong specificity. The specific scheme is as follows.

[0034] In the first aspect, the embodiments of the present application provide a primer and a probe. Specifically, the embodiments of the present application provide a primer and a probe combination for detecting Talaromyces marneffei based on droplet digital PCR, including a pair of primer sequences and a probe sequence. The primer and the probe can specifically bind to the ITS gene of Talaromyces marneffei, and the specific sequence is as follows.

[0035] The upstream primer sequence is SEQ NO.1: 5′-CTGTCCGAGCGTCATTTC-3′;

[0036] The downstream primer sequence is SEQ NO.2: 5′-AGGTCAACCGTGGTAAATATG-3′;

[0037] The probe sequence is SEQ NO. 3: 5'-CTTGTGTGTTGGGTGTGGTCCCTC-3'.

[0038] The embodiments of the present application use the above primers and probes to quickly and accurately detect Talaromyces marneffei based on droplet digital PCR, which not only requires a small amount of sample to be tested, does not require pathogenic culture, and is time-consuming, but also has no cross-reaction to other pathogens, has good specificity, and high sensitivity. Therefore, it has important application value for the early diagnosis and treatment of Talaromyces marneffei.

[0039] In some embodiments, the final concentrations of primers and probes for detecting Talaromyces marneffei are: 0.25-1.0 μM for the upstream primer, 0.25-1.0 μM for the downstream primer, and 0.10-0.40 μM for the probe.

[0040] Furthermore, the final concentrations of primers and probes used to detect Talaromyces marneffei are: 0.5 μM for upstream primers, 0.5 μM for downstream primers, and 0.25 μM for probes. In the embodiment of the present application, based on the droplet digital PCR reaction system, four gradients are set for the final concentrations of upstream and downstream primers, which are 0.25 μM, 0.5 μM, 0.75 μM, and 1.0 μM, respectively. The results showed that the final concentration of the upstream and downstream primers was 0.5 μM, and the distinction between positive droplets and negative droplets was most obvious. And further combined with the optimized probe concentration, in the final droplet digital PCR reaction system, the final concentration of the upstream primer was set to 0.5 μM, the final concentration of the downstream primer was set to 0.5 μM, and the final concentration of the probe was set to 0.25 μM. Such a droplet digital PCR detection effect is better.

[0041] In some embodiments, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group. Specifically, the fluorescent reporter group includes at least one of FAM, VIC, HEX, TET, JOE, CY3 and CY5; the fluorescent quencher group includes at least one of TAMARA, MGB, BHQ-1, BHQ-2 and BHQ-3. Further, the 5' end of the probe is labeled with a FAM fluorescent reporter group, and the 3' end is labeled with a BHQ-1 fluorescent quencher group.

[0042] In the second aspect, the embodiments of the present application provide an application. Specifically, the primers and probes provided in the first aspect of the embodiments of the present application can quickly, accurately and sensitively detect Talaromyces marneffei based on droplet digital PCR, so the primers and probes can be used to prepare a kit for detecting Talaromyces marneffei based on droplet digital PCR.

[0043] In a third aspect, an embodiment of the present application provides a kit for detecting Talaromyces marneffei. The kit of the embodiment of the present application includes the primers and probes provided in the first aspect of the embodiment of the present application. Based on the characteristics of the primers and probes, the kit can quickly, accurately and sensitively detect Talaromyces marneffei pathogens based on droplet digital PCR, providing important value for achieving early diagnosis of Talaromyces marneffei.

[0044] In some embodiments, the kit detects Talaromyces marneffei based on droplet digital PCR.

[0045] In some embodiments, the kit further includes: droplet digital PCR system reagents. For example, the droplet digital PCR system reagents include droplet digital PCR amplification enzymes, which can specifically refer to the reagents in 4×ProbedPCRUniMix (Cat. No. 23013) of Xinyi Biotechnology Co., Ltd.

[0046] In some embodiments, the kit further comprises a sample nucleic acid extraction reagent. For example, the sample nucleic acid extraction reagent comprises QIAamp UCP Pathogen Mini Kit (Qiagen, 50214).

[0047] The present application embodiment provides a detection method for detecting Talaromyces marneffei based on droplet digital PCR based on the above-mentioned kit, comprising the following steps:

[0048] (1) Extract nucleic acid from samples. The samples may be whole blood, plasma, serum, bone marrow, cerebrospinal fluid, urine, bronchoalveolar lavage fluid, biopsy tissue or other samples from other sources.

[0049] (2) Mixing the sample nucleic acid with the primers and probes of the present invention and the droplet digital PCR system reagents to obtain a droplet digital PCR reaction system.

[0050] (3) The droplet digital PCR reaction system is transferred to a droplet generation chip, and then placed in a droplet preparation instrument to generate droplets.

[0051] (4) After droplet generation, PCR amplification is performed.

[0052] (5) The PCR amplification product is placed in a droplet detection chip, and then placed in a droplet detector for fluorescence signal reading and analysis to obtain the copy number of the target gene.

[0053] In some embodiments, in the droplet digital PCR reaction system of step (2), the final concentration of the upstream primer is 0.5 μM, the final concentration of the downstream primer is 0.5 μM, and the final concentration of the probe is 0.25 μM.

[0054] In some embodiments, the PCR amplification program of step (4) is 95° C. pre-denaturation for 2 min; 94° C. denaturation for 30 sec; 59° C. annealing and extension for 1 min, 40 cycles. The heating and cooling speed of each step is 1.5° C. / s.

[0055] In some embodiments, in the above-mentioned droplet digital PCR reaction step, the annealing temperature is set to 4 gradients, namely 58°C, 59°C, 60°C, and 61°C. The results show that when the annealing temperature is 59°C, the positive droplets are most clearly distinguished from the negative droplets. Therefore, when the primers and probes of the embodiments of the present application are used to detect the pathogen of Talaromyces marneffei based on droplet digital PCR, the primers and probes are combined with a certain final concentration (the final concentration of the upstream primer is 0.5 μM, the final concentration of the downstream primer is 0.5 μM, and the final concentration of the probe is 0.25 μM) and the annealing temperature of 59°C can better detect Talaromyces marneffei.

[0056] In some embodiments, the kit further includes a positive control and a negative control.

[0057] In short, the embodiments of the present application do not require etiological culture, and the time consumption is short, and the detection can be completed within half a day. The required sample dosage is small, and samples such as peripheral blood, bone marrow, cerebrospinal fluid, urine, alveolar lavage fluid, and biopsy tissue can be detected. Based on the primers and probes of the embodiments of the present application, Talaromyces marneffei can be specifically detected, and there is no cross reaction to other pathogens, and the specificity is good. Compared with fluorescent quantitative PCR, the embodiments of the present application can achieve absolute quantification without relying on a standard curve, and the sensitivity is higher, which has important application value for the early diagnosis and treatment of Talaromyces marneffei.

[0058] The following describes the invention in conjunction with specific embodiments.

[0059] Example 1 Design and screening of primers and probes

[0060] ITS (Internal Transcribed Spacer) is the internal transcribed spacer in fungal ribosomal DNA. Due to the high variability of ITS among different species and the stability within species, this example compares and analyzes the ITS genes of various strains of T. marneffei, selects the conservative region sequence to design 4 sets of primers and probe sequences (see combination 1-4 in Table 1), and searches the literature and patents to obtain the existing 2 sets of ITS primers and probe sequences (see combination 5-6 in Table 1).

[0061] Then the fluorescence quantitative PCR experiment found that Figure 1 and Figure 2 As shown (the red curve is the primer of combination 1, and the green curves are the primers of combinations 2-6 respectively). When the same amount of template is input, the reaction of the primer of combination 1 takes precedence, and the melting curve is a single peak, without nonspecific amplification, indicating that the amplification performance and specificity of combination 1 are better than other combinations. Therefore, this application selects combination 1 for droplet digital PCR detection of Talaromyces marneffei.

[0062] Table 1 ITS primers and probe sequences of Talaromyces marneffei

[0063]

[0064] Example 2 Establishment of droplet digital PCR system

[0065] Using the primers and probes of combination 1 in Example 1, the 5' end of the probe is labeled with a FAM fluorescent reporter group, and the 3' end is labeled with a BHQ-1 fluorescent quencher group, a droplet digital PCR method for detecting Talaromyces marneffei is established, and the steps are as follows:

[0066] (1) Sample nucleic acid extraction

[0067] Talaromyces marneffei and other pathogens (Cryptococcus neoformans, Candida albicans, Aspergillus flavus, Mycobacterium tuberculosis, Staphylococcus aureus, Salmonella enteritidis, Pseudomonas aeruginosa, Klebsiella pneumoniae) were cultured, and nucleic acids were extracted using QIAamp UCPPathogen Mini Kit (Qiagen, 50214), and finally eluted with 50 μL TE buffer to obtain nucleic acids.

[0068] (2) Preparation of droplet digital PCR reaction system

[0069] The 4×Probe dPCRUniMix (Cat. No. 23013) kit of Xinyi Biotechnology Co., Ltd. was used to prepare the droplet digital PCR reaction system according to Table 2. The final concentration of the upstream primer was 0.5 μM, the final concentration of the downstream primer was 0.5 μM, and the final concentration of the probe was 0.25 μM.

[0070] Table 2 Droplet digital PCR reaction system

[0071] Components volume 4×Probe dPCR Mix 7.5μL Upstream primer (10 μM) 1.5μL Downstream primer (10 μM) 1.5μL Probe (10 μM) 0.75μL Sample nucleic acid 5μL Sterile water 13.75μL Total volume 30μL

[0072] (3) The PCR reaction system is transferred to a droplet generation chip, and then placed in a droplet preparation instrument to generate droplets.

[0073] (4) After droplet generation, PCR amplification was performed with the following reaction procedure: pre-denaturation at 95°C for 2 min, denaturation at 94°C for 30 sec, annealing and extension at 59°C for 1 min, and 40 cycles.

[0074] (5) The PCR amplification product is placed in a droplet detection chip, and then placed in a droplet detector for fluorescence signal reading and analysis to obtain the copy number of the target gene.

[0075] The experimental results are as follows Figure 3 As shown, no positive droplets were detected for 8 pathogens including Cryptococcus neoformans, Candida albicans, Aspergillus flavus, Mycobacterium tuberculosis, Staphylococcus aureus, Salmonella enteritidis, Pseudomonas aeruginosa, and Klebsiella pneumoniae, while positive droplets were detected for Talaromyces marneffei, indicating that the embodiments of the present application can specifically detect Talaromyces marneffei.

[0076] Example 3 Plasma clinical sample detection

[0077] The sample in this embodiment is a human plasma sample. Peripheral venous blood was drawn from 119 patients with AIDS and Talaromycosis marneffei and 47 patients with non-Talaromycosis marneffei (28 patients with AIDS and other opportunistic infections, 13 patients with simple AIDS, and 6 normal persons) using a vacuum blood collection tube containing an anticoagulant, and the blood collection tube was placed in a centrifuge, centrifuged at 3000rpm for 5min, 400μL of the upper plasma was taken, and nucleic acid was extracted using QIAamp UCP Pathogen Mini Kit (Qiagen, 50214), and finally 50μL TE buffer was used to elute the nucleic acid, and a total of 166 plasma sample nucleic acids were obtained.

[0078] The droplet digital PCR detection was performed according to the steps of Example 2, and the same primers and probes were used for fluorescence quantitative PCR detection. The fluorescence quantitative PCR reaction system was prepared according to Table 3. Among them, the final concentration of the upstream primer was 0.25 μM, the final concentration of the downstream primer was 0.25 μM, and the final concentration of the probe was 0.25 μM. The fluorescence quantitative PCR reaction steps were: 95°C pre-denaturation for 20 seconds; 95°C denaturation for 3 seconds, 59°C annealing and extension for 30 seconds, and 40 cycles.

[0079] Table 3 Fluorescence quantitative PCR reaction system

[0080]

[0081] The experimental results are shown in Table 4. Among the 119 plasma samples of AIDS patients with Talaromycosis marneffei, 110 were detected positive by droplet digital PCR, with a positive detection rate of 92.44%; while 104 were detected positive by fluorescent quantitative PCR, with a positive detection rate of 87.39%. For the 47 plasma samples of patients who were not infected with Talaromycosis marneffei, the results of both droplet digital PCR and fluorescent quantitative PCR were negative. It can be seen that the method provided in the present application for detecting Talaromyces marneffei in human plasma based on droplet digital PCR has good specificity, a higher detection positivity rate and higher sensitivity than fluorescent quantitative PCR.

[0082] Table 4 Test results of human plasma samples

[0083]

[0084] Example 4 Whole blood clinical sample detection

[0085] The sample in this embodiment is a human whole blood sample. Peripheral venous blood was drawn from 3 patients with AIDS and Talaromycosis marneffei and 3 patients with AIDS and other opportunistic infections using a vacuum blood collection tube containing an anticoagulant, 400 μL of whole blood was taken, and nucleic acid was extracted using QIAamp UCP Pathogen Mini Kit (Qiagen, 50214), and finally 50 μL of TE buffer was used to elute the nucleic acid. The remaining steps were the same as in Example 2.

[0086] The experimental results are shown in Table 5. The copy numbers of droplet digital PCR detection of whole blood samples from 3 patients with AIDS and Talaromycosis marneffei were all greater than 0, and the results were all positive; the copy numbers of droplet digital PCR detection of whole blood samples from 3 patients with AIDS and other opportunistic infections (non-Talaromycosis marneffei patients) were all 0, and the results were all negative. It can be seen that the droplet digital PCR method provided in this application can detect Talaromyces marneffei in human whole blood with high sensitivity and good specificity.

[0087] Table 5 Human whole blood sample test results

[0088]

[0089] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A set of primers and probes for detecting Talaromyces marneffei, characterized in that: include: Upstream primer, the nucleotide sequence is shown in SEQ ID NO.1; Downstream primer, nucleotide sequence is shown in SEQ ID NO.2; and a probe, wherein the nucleotide sequence is shown in SEQ ID NO.

3.

2. The primer and probe according to claim 1, characterized in that The final concentrations of the primers and probes when used to detect Talaromyces marneffei are: 0.25-1.0 μM for the upstream primer, 0.25-1.0 μM for the downstream primer, and 0.10-0.40 μM for the probe.

3. The primer and probe according to claim 2, characterized in that: The final concentrations of the primers and probes used for detecting Talaromyces marneffei are: 0.5 μM for the upstream primer, 0.5 μM for the downstream primer, and 0.25 μM for the probe.

4. The primer and probe according to any one of claims 1 to 3, characterized in that: The 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group.

5. The primer and probe according to claim 4, characterized in that: The fluorescent reporter group includes at least one of FAM, VIC, HEX, TET, JOE, CY3 and CY5; And / or, the fluorescence quenching group includes at least one of TAMARA, MGB, BHQ-1, BHQ-2 and BHQ-3.

6. Use of the primers and probes described in any one of claims 1 to 5 in the preparation of a kit for detecting Talaromyces marneffei based on droplet digital PCR.

7. A kit for detecting Talaromyces marneffei, characterized in that: The kit comprises the primers and probes according to any one of claims 1 to 5.

8. The kit according to claim 7, characterized in that The kit is based on droplet digital PCR to detect Talaromyces marneffei.

9. The kit according to claim 7, characterized in that The kit also includes: droplet digital PCR system reagents.

10. The kit according to any one of claims 7 to 9, characterized in that The kit also includes a sample nucleic acid extraction reagent.

Images