Primer probe combination and kit for aspergillus subspecies detection and detection method and application of primer probe combination and kit

Abstract

The invention provides a primer probe combination, a kit, a detection method and application for aspergillus subspecies detection, and belongs to the technical field of biology. The primer probe combination for aspergillus subspecies detection comprises specific primers and probes aiming at aspergillus fumigatus, aspergillus flavus, aspergillus terreus and aspergillus niger, the nucleotide sequences of the specific primers and the probes are shown as SEQ ID NO.1-SEQ ID NO.12, and the primer probe combination further comprises specific primers and probes of internal reference, and the nucleotide sequences of the specific primers and the probes are shown as SEQ ID NO.13-SEQ ID NO.15. The method has the advantages of high sensitivity, high specificity and multi-target detection; compared with the tedious steps of gel electrophoresis verification required by common PCR, the method has the advantages of simplicity, convenience and rapidness in operation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a primer-probe combination, a kit and a detection method and application thereof for detecting Aspergillus species. Background Art

[0002] Aspergillus is a type of fungus that is widely distributed in the environment, such as soil, air, and various organic surfaces. They can reproduce in large numbers under suitable conditions. Some Aspergillus species, such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, and Aspergillus niger, are potential hazards to human health and are common pathogenic fungi in clinical practice.

[0003] The incidence of invasive aspergillosis (including chronic pulmonary aspergillosis and invasive pulmonary aspergillosis) is on the rise worldwide. According to statistics, the number of cases of invasive aspergillosis worldwide exceeds 300,000 each year. However, due to the limitations of current diagnostic technology, the actual number of confirmed cases may only account for 50% to 65% of all cases. Even if patients can receive timely diagnosis and treatment, their mortality rate is still high, reaching 30% to 80%. This severe situation highlights the importance and urgency of accurate and rapid diagnosis of Aspergillus infection.

[0004] Aspergillus fumigatus is the main pathogen of invasive aspergillosis, and the number of clinically infected cases caused is large and the condition is relatively serious. Its spores are extremely small and can remain suspended in the air for a long time, making them easily inhaled into the human respiratory tract, thereby invading the lung tissue and triggering a series of inflammatory reactions and pathological changes. Aspergillus fumigatus has attracted much attention because it can produce aflatoxin, a highly carcinogenic toxin. It can not only cause food contamination, but also, under certain conditions, human inhalation or contact with substances containing Aspergillus fumigatus may cause infection, especially for people with weakened immune function. Aspergillus terreus and Aspergillus niger can also cause human infections under suitable conditions, such as lung infections, sinusitis, etc., posing a threat to the health of patients.

[0005] At present, the traditional clinical diagnosis method of invasive aspergillosis mainly relies on fungal culture, histopathological examination, etc. Although fungal culture is the gold standard for diagnosis, it has obvious defects. It is extremely time-consuming and usually takes several days or even weeks to obtain results, which will undoubtedly delay timely clinical diagnosis and treatment, resulting in further deterioration of the patient's condition. Although histopathological examination can directly observe the fungal morphology in the tissue, it has high requirements for sample collection and processing, and is invasive to a certain extent, which may bring additional pain and risks to patients. In addition, antigen-antibody detection methods are also used to a certain extent in the diagnosis of Aspergillus infection. However, this method has the problem of high false positive rate, which is easy to cause misdiagnosis and interfere with clinical decision-making. At the same time, the preparation of specific antibodies is relatively difficult, which limits its wide application in clinical diagnosis. Therefore, it is urgent to develop a rapid, accurate, highly specific and easy-to-operate Aspergillus detection method to meet the needs of clinical diagnosis and disease prevention and control. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a primer-probe combination, a kit and a detection method and application thereof for detecting Aspergillus species, which has the advantages of high sensitivity, high specificity and multi-target detection. The primer-probe combination includes one or more of the following primers and probes:

[0007] The nucleotide sequences of the specific primers for Aspergillus niger are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is shown in SEQ ID NO.3;

[0008] The nucleotide sequences of the specific primers for Aspergillus terreus are shown in SEQ ID NO.4 and SEQ ID NO.5, and the nucleotide sequence of the probe is shown in SEQ ID NO.6;

[0009] The nucleotide sequences of the specific primers for Aspergillus fumigatus are shown in SEQ ID NO.7 and SEQ ID NO.8, and the nucleotide sequence of the probe is shown in SEQ ID NO.9;

[0010] The nucleotide sequences of the specific primers for Aspergillus flavus are shown in SEQ ID NO.10 and SEQ ID NO.11, and the nucleotide sequence of the probe is shown in SEQ ID NO.12.

[0011] Preferably, specific primers and probes for internal references are also included; the internal reference is Bacillus subtilis, the nucleotide sequences of the specific primers are shown in SEQ ID NO.13 and SEQ ID NO.14, and the nucleotide sequence of the probe is shown in SEQ ID NO.15.

[0012] More preferably, the detection probes of Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Aspergillus flavus and the internal reference are modified with a fluorescent group at the 5' end and a quenching group at the 3' end;

[0013] The fluorescent group is selected from any one of FAM, VIC, TET, JOE, HEX, CY3, CY5, TAMRA, ROX, Texas Red, CY5.5 or CY7;

[0014] The quenching group is selected from any one of BHQ0, BHQ1, BHQ2, BHQ3 or MGB.

[0015] The present invention also provides a kit for detecting Aspergillus species, which comprises the primer-probe combination.

[0016] The present invention also provides a method for detecting Aspergillus species using the kit, comprising the following steps: (1) extracting nucleic acid from a sample to be tested; (2) performing qPCR amplification using the extracted nucleic acid as a template, interpreting the result of the qPCR amplification, and identifying the Aspergillus in the sample to be tested.

[0017] Preferably, the reaction system of the qPCR amplification is: 0.1 to 50 μL of template, 5 to 50 μL of qPCR amplification reagent;

[0018] The qPCR amplification reagent is prepared as follows: 2×PCR Mix 1×; 0.01 to 10 μM of each specific primer and 0.01 to 10 μM of each probe.

[0019] Preferably, the qPCR amplification program is: pre-denaturation at 95°C for 0.1 to 10 min; denaturation at 95°C for 0.1 to 30 s, annealing and extension at 55 to 65°C for 3 to 120 s, 30 to 70 cycles, and collecting fluorescence signals during the annealing and extension phase of each cycle.

[0020] Preferably, the criteria for judging the result of the qPCR amplification as positive are: (1) the internal reference Ct value is ≤40.0; (2) the target Ct value is ≤40.0; (3) the amplification curve presents a standard "S"-shaped curve.

[0021] The present invention also provides the use of the primer-probe combination or the kit in detecting Aspergillus species or preparing an Aspergillus species detection reagent.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a primer-probe combination, a kit and a detection method for detecting Aspergillus species based on a real-time fluorescence quantitative PCR method, and diagnoses Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus niger, which are common Aspergillus species. The technical solution of the present invention has the advantages of high sensitivity (the detection rate of 100 copies / mL of four targets is ≥95%), high specificity (there is no cross-reaction between the method and other pathogens) and multi-target detection (simultaneous detection of four Aspergillus species). Compared with the cumbersome steps of gel electrophoresis verification required for ordinary PCR, the present invention has the advantages of simple and rapid operation. DETAILED DESCRIPTION

[0024] The present invention provides a primer-probe combination for detecting Aspergillus species, including at least one specific primer and probe for Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus niger. Preferably, it also includes a specific primer and probe for an internal reference, and the internal reference is Bacillus subtilis. More preferably, the 5' end of the detection probe of Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Aspergillus fumigatus and the internal reference is modified with a fluorescent group, and the 3' end is modified with a quenching group, and the fluorescent group is selected from any one of FAM, VIC, TET, JOE, HEX, CY3, CY5, TAMRA, ROX, Texas Red, CY5.5 or CY7, and the quenching group is selected from any one of BHQ0, BHQ1, BHQ2, BHQ3 or MGB. As shown in Table 1:

[0025] Table 1 Primer probe combinations

[0026]

[0027]

[0028] The present invention also provides a kit for detecting Aspergillus species, comprising the above primer-probe combination. Preferably, the kit comprises a nucleic acid extraction reagent, a qPCR amplification reagent and an internal reference; further preferably, 2×PCRMix1×, 0.01 to 10 μM each of the specific primers, and 0.01 to 10 μM each of the probes; more preferably, the primer-probe combination shown in Table 1 is used to prepare the qPCR amplification reagents for Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus niger, and each component is prepared according to Table 2:

[0029] Table 2 Primer probe concentration settings in qPCR amplification reagents

[0030]

[0031]

[0032] The present invention also provides a method for detecting Aspergillus species using the above kit, comprising the following steps:

[0033] (1) Extracting nucleic acid from the sample to be tested as a template. As an implementation method, the genomic DNA extraction kit (DP307) of Tiangen is used for extraction and amplification according to the instructions.

[0034] (2) Prepare the qPCR reaction system. Preferably, the template is 0.1 to 50 μL and the qPCR amplification reagent is 5 to 50 μL; more preferably, the template is 20 μL and the qPCR amplification reagent is 30 μL (Table 2).

[0035] (3) Perform qPCR amplification. The preferred qPCR amplification program is: pre-denaturation at 95°C for 0.1 to 10 min; denaturation at 95°C for 0.1 to 30 s, annealing and extension at 55 to 65°C for 3 to 120 s, 30 to 70 cycles, and collecting fluorescence signals during the annealing and extension phase of each cycle; further preferred is pre-denaturation at 95°C for 5 min; denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, 40 cycles, and collecting fluorescence signals during the annealing and extension phase of each cycle. As an implementable method, the PCR eight-row array is placed in the sample slot of the fluorescent quantitative PCR instrument, and after the amplification program is set, amplification is started.

[0036] (4) Interpret the qPCR results and identify the Aspergillus in the sample to be tested. The preferred criteria for interpreting the qPCR results as positive are: (1) the internal reference Ct value is ≤40.0, and the negative control group and the no-template control group have no Ct value; if it does not meet the criteria, perform qPCR again, or re-extract nucleic acid for qPCR; (2) the pathogen Ct value is ≤40.0; (3) the amplification curve presents a standard "S" curve. It is preferred that different bacterial species correspond to different fluorescent signals, and the specific bacterial species is determined based on the fluorescent channel of the detection signal.

[0037] The present invention also provides the use of the primer-probe combination or the kit in detecting Aspergillus species or preparing an Aspergillus species detection reagent.

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] Example 1

[0040] The detection sensitivity test of the primer-probe combination of the present invention.

[0041] Add 1×10 6 copies / mL, 1×10 5 copies / mL, 1×10 4 copies / mL, 1×10 3 copies / mL, 1×10 2The cultures of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus with 10 copies / mL were extracted and amplified using the Tiangen genomic DNA extraction kit (DP307) according to the instructions.

[0042] The extracted nucleic acids of the four pathogens are used as templates for amplification. After the amplification system is prepared, mix well and load onto the machine.

[0043] Table 3 Amplification system

[0044] Element Reaction system - 50 μL PrimerMix (primer probe combination) 3μL 2×PCRMix 25μL Internal reference primer probe Mix 0.5μL water 1.5μL template 20μL

[0045] Place the eight-strip PCR array in the sample slot of the fluorescent quantitative PCR instrument, and start amplification after setting up the amplification program.

[0046] Table 4 Amplification procedures

[0047]

[0048] Test results:

[0049] As shown in Tables 5-8, the detection sensitivity of the primer-probe combination of the present invention to four types of Aspergillus can reach 100 copies / mL.

[0050] Table 5 Detection results of Aspergillus fumigatus

[0051]

[0052] Table 6 Detection results of Aspergillus flavus

[0053]

[0054]

[0055] Table 7 Detection results of Aspergillus terreus

[0056]

[0057] Table 8 Detection results of Aspergillus niger

[0058]

[0059] Example 2

[0060] The detection specificity test of the primer-probe combination of the present invention is specifically implemented in the following steps:

[0061] 1. Use negative sputum as negative matrix and add cross-reactants to the sputum to make the concentration of virus cross-reactants 10 5 PFU / mL, bacterial cross-reactant concentration is 10 6 CFU / mL and use it as the sample to be tested.

[0062] 2. Nucleic acid extraction

[0063] The Tiangen genomic DNA extraction kit (DP307) was used for extraction and amplification according to the instructions.

[0064] 3. Preparation of qPCR amplification system:

[0065] After the amplification system is prepared, mix well and load onto the machine. The reaction system is as follows:

[0066] Table 9 Reaction system

[0067] Element Reaction system - 60 μL PrimerMix (primer probe combination) 3uL 2×PCRMix 25uL Internal reference primer probe Mix 0.5uL water 11.5uL template 20uL

[0068] Place the eight-strip PCR array in the sample slot of the fluorescent quantitative PCR instrument, and start amplification after setting up the amplification program.

[0069] Table 10 Amplification program

[0070]

[0071] Test results:

[0072] As shown in Table 11, the combined detection kit for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus had no cross-reaction with the 29 cross-reactants in the table and had good specificity.

[0073] Table 11 Specificity test results

[0074]

[0075]

[0076]

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer-probe combination for detecting Aspergillus species, characterized in that: Include one or more of the following primers and probes: The nucleotide sequences of the specific primers for Aspergillus niger are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is shown in SEQ ID NO.3; The nucleotide sequences of the specific primers for Aspergillus terreus are shown in SEQ ID NO.4 and SEQ ID NO.5, and the nucleotide sequence of the probe is shown in SEQ ID NO.6; The nucleotide sequences of the specific primers for Aspergillus fumigatus are shown in SEQ ID NO.7 and SEQ ID NO.8, and the nucleotide sequence of the probe is shown in SEQ ID NO.9; The nucleotide sequences of the specific primers for Aspergillus flavus are shown in SEQ ID NO.10 and SEQ ID NO.11, and the nucleotide sequence of the probe is shown in SEQ ID NO.

12.

2. The primer-probe combination according to claim 1, characterized in that: It also includes specific primers and probes for internal references; the internal reference is Bacillus subtilis, the nucleotide sequences of the specific primers are shown in SEQ ID NO.13 and SEQ ID NO.14, and the nucleotide sequence of the probe is shown in SEQ ID NO.

15.

3. The primer-probe combination according to claim 1 or 2, characterized in that: The detection probes of Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Aspergillus flavus and internal reference are modified with a fluorescent group at the 5' end and a quenching group at the 3' end; The fluorescent group is selected from any one of FAM, VIC, TET, JOE, HEX, CY3, CY5, TAMRA, ROX, Texas Red, CY5.5 or CY7; The quenching group is selected from any one of BHQ0, BHQ1, BHQ2, BHQ3 or MGB.

4. A kit for detecting Aspergillus species, characterized in that: The kit comprises the primer-probe combination according to any one of claims 1 to 3.

5. A method for detecting Aspergillus species using the kit according to claim 4, characterized in that: The following steps are involved: (1) extracting nucleic acid from the sample to be tested; (2) performing qPCR amplification using the extracted nucleic acid as a template, interpreting the results of the qPCR amplification, and identifying the Aspergillus in the sample to be tested.

6. The method for detecting Aspergillus species according to claim 5, characterized in that: The reaction system of the qPCR amplification is: 0.1 to 50 μL of template and 5 to 50 μL of qPCR amplification reagent; The qPCR amplification reagent is prepared as follows: 2×PCR Mix 1×; 0.01 to 10 μM of each of the specific primers described in claim 1 and claim 2, and 0.01 to 10 μM of each of the probes.

7. The method for detecting Aspergillus species according to claim 5, characterized in that: The qPCR amplification procedure is: pre-denaturation at 95°C for 0.1 to 10 min; denaturation at 95°C for 0.1 to 30 s, annealing and extension at 55 to 65°C for 3 to 120 s, 30 to 70 cycles, and collecting fluorescence signals during the annealing and extension phase of each cycle.

8. The method for detecting Aspergillus species according to claim 5, characterized in that: The criteria for judging the qPCR amplification result as positive are: (1) the internal reference Ct value is ≤40.0; (2) the target Ct value is ≤40.0; (3) the amplification curve presents a standard "S" curve.

9. Use of the primer-probe combination according to any one of claims 1 to 3 or the kit according to claim 4 in detecting Aspergillus species or preparing an Aspergillus species detection reagent.

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