RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit for simultaneously detecting multiple human herpes viruses and use method thereof

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to an RT-PCR kit for simultaneously detecting multiple human herpes viruses and a using method thereof.The kit comprises RT-PCR primer probes, a buffer solution and Taq enzyme, the primer probes and the buffer solution are fully premixed for loading the Taq enzyme, and the primer probes and the buffer solution are mixed for loading the Taq enzyme. The primer probes comprise forward and reverse amplification primers and detection probes respectively aiming at the human herpes virus type 5, the human herpes virus type 6, the human herpes virus type 7 and the human herpes virus type 8, and the sequences are respectively shown as SEQ ID NO.1-SEQ ID NO.13. The kit disclosed by the invention can be used for rapidly detecting and identifying the four types of human herpes viruses at the same time; the kit has high accuracy, specificity and sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and specifically relates to an RT-PCR kit for simultaneously detecting multiple human herpes viruses and a use method thereof. Background Art

[0002] Herpes virus infection is a common viral disease caused by a variety of herpes viruses, including herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), etc. These viruses are highly contagious and potentially pathogenic, and can cause a variety of clinical manifestations ranging from mild skin lesions to severe neurological diseases. Herpes virus infection can be divided into primary infection and latent infection. Primary infection usually occurs in the early life of an individual, while latent infection is after primary infection, the virus remains dormant in certain cells in the body until the body's immunity decreases or other triggering factors appear, the virus is reactivated, leading to recurrent infection. Early diagnosis is of great significance for herpes virus infection, which can not only improve the treatment effect, reduce the misdiagnosis rate and the risk of complications, but also improve the quality of life, promote disease management, and improve diagnostic accuracy.

[0003] Human herpesvirus type 5 (HHV-5), also known as human cytomegalovirus (HCMV), belongs to the betaherpesvirus subfamily of the herpesviridae family. This virus is widespread worldwide and is particularly common in immunocompetent individuals. Infection with HCMV does not usually cause symptoms, but in individuals with compromised immune systems, such as AIDS patients, organ transplant recipients, and patients undergoing bone marrow transplants, it can cause severe illness.

[0004] Human herpesvirus type 6 (HHV-6) is a double-stranded DNA virus belonging to the β-herpesvirus subfamily. It was first isolated from monocytes in the peripheral blood of AIDS patients and patients with lymphoproliferative disorders. HHV-6 has significant lymphotropism and neurotropism and is associated with a variety of diseases, including infantile exanthema and infection after organ transplantation.

[0005] Human herpesvirus 7 (HHV-7) is a lymphotropic virus belonging to the β-herpesvirus subfamily and was first isolated in 1990. HHV-7 shares CD4 as its receptor with human immunodeficiency virus (HIV), indicating that HHV-7 is able to infect T cells and plays an important role in the immune system. Primary infection with HHV-7 usually occurs in childhood, is mainly transmitted through saliva, and HHV-7 can be frequently isolated in the saliva of healthy adults.

[0006] Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated human herpesvirus (KSHV), is a gamma-herpesvirus associated with a variety of diseases, especially lymphocyte-related malignancies. HHV-8 infection is associated with diseases such as Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD).

[0007] At present, the detection method of human herpes virus clinically is mainly cell culture and serological method.Cell culture method has shortcomings such as slow speed, low success rate, high cost and large labor intensity.Immunological method is the current mainstream detection method, but detection sensitivity and specificity can not meet clinical needs.Based on the fluorescent PCR nucleic acid detection technology, there is an outstanding advantage in time, flux, sensitivity and specificity, and it is more and more widely used.There is a need for primers and probes that can more efficiently realize the multi-index detection of human herpes virus. Summary of the invention

[0008] In view of the problems of insufficient sensitivity and poor specificity in the existing fluorescent PCR technology for detecting human herpes virus mentioned in the background technology, in order to solve the above problems, the present invention discloses an RT-PCR kit for simultaneously detecting multiple human herpes viruses and a method for using the kit, by specifically designing primers and probes, thereby improving the sensitivity and specificity of detection.

[0009] The technical solution of the present invention is as follows:

[0010] A RT-PCR kit for simultaneously detecting multiple human herpes viruses. The kit is a fully premixed reagent, comprising a buffer solution for amplification, a taq enzyme, and a primer probe for detection. The human herpes viruses detected by the kit include human herpes virus type 5, human herpes virus type 6, human herpes virus type 7, and human herpes virus type 8.

[0011] Further, the primers include: a forward primer for detecting human herpesvirus type 5:

[0012] 5'-GGCCGTTACTGTCTGCAGGA-3', as shown in SEQ ID No. 1, reverse amplification primer: 5'-GGCCTCGTAGTGAAAATTAATGGT-3', as shown in SEQ ID No. 2;

[0013] Forward primer for detection of human herpesvirus type 6:

[0014] 5'-AAAGACCTAAATTGCCGCTACCT-3', as shown in SEQ ID No. 3,

[0015] Reverse amplification primer: 5'-GCAAGCTCATGAACATCGTCA-3', as shown in SEQ ID No. 4;

[0016] Forward primer for detection of human herpesvirus type 7:

[0017] 5'-CCAATCCTTCCGAAACCGAT-3', as shown in SEQ ID No.5,

[0018] Reverse amplification primer: 5'-CGGAAGTCACTGGAGTAATGACAA-3', as shown in SEQ ID No.6;

[0019] Forward primer for detection of human herpesvirus type 8:

[0020] 5'-CTGCGACATATTTCCCTGATCC-3', as shown in SEQ ID No.7,

[0021] Reverse amplification primer: 5'-TGGATCATGACTACGCTCACCAT-3', as shown in SEQ ID No.8.

[0022] Further, the probe includes: a probe for human herpes virus type 5:

[0023] 5'-CCGTATTGGTGCGCGATCTGTTCAA-3', as shown in SEQ ID No. 9;

[0024] Probes for human herpesvirus type 6:

[0025] 5'-TTAGATGGTGGTGAGCTGGGATCGGT-3', as shown in SEQ ID No. 10;

[0026] Probe for human herpesvirus type 7: 5'-CTCGCAGATTGCTTGTTGGCCATG-3', as shown in SEQ ID No. 11; 5'-TCGGTAAGATAAATAAGCAATCA-3', as shown in SEQ ID No. 12;

[0027] Probe for human herpesvirus type 8: 5'-TTCATGCGAGCCACCAG-3', as shown in SEQ ID No.13.

[0028] Furthermore, the 5' end of the human herpes virus type 5 detection probe sequence is labeled with a FAM fluorescent group, and the 3' end is labeled with a fluorescent quenching group MGB;

[0029] The 5' end of the human herpes virus type 6 detection probe sequence is labeled with a VIC fluorescent group, and the 3' end is labeled with a fluorescent quenching group MGB;

[0030] The 5' end of the human herpes virus 7 detection probe sequence is labeled with a CY5 fluorescent group, and the 3' end is labeled with a fluorescent quenching group BHQ2;

[0031] The 5' end of the human herpes virus type 8 detection probe sequence is labeled with a ROX fluorescent group, and the 3' end is labeled with a fluorescent quenching group BHQ2.

[0032] Furthermore, the bases "A+", "C+" and "G+" in the primer sequence are locked nucleic acid modified bases.

[0033] Furthermore, the final concentration of the primers in the reagent is 444 nM, and the final concentration of the probe is 222 nM.

[0034] The present invention also provides a multiple rapid RT-PCR method for simultaneously detecting nucleic acids of human herpesvirus types 5, 6, 7, and 8, the specific steps of which include:

[0035] Step 1: Extract sample nucleic acid;

[0036] Step 2: Thaw and mix the reagents and then dispense them directly into 96-well plates or eight-well tubes. Add the extracted sample template, negative control, and positive quality control products into different PCR reaction tubes, cover the tubes, and place them into the fluorescence quantitative PCR instrument for fluorescence PCR detection.

[0037] Step 3: After the reaction in step 2 is completed, set the automatic baseline adjustment and analyze the test results according to the amplification curve and Ct value.

[0038] Furthermore, the reaction conditions are: 95° C., 2 min in the pre-denaturation stage; 95° C., 5 s, 56° C., 30 s in the amplification stage, and 40 cycles.

[0039] Compared with the prior art, the kit of the present invention has the following advantages:

[0040] (1) The present invention realizes the typing detection and identification of 4 kinds of human herpes viruses, can accurately determine the specific pathogens, guide further treatment, and improve the scientificity and effectiveness of the treatment plan. The identification of specific pathogens helps to statistically analyze the types of pathogens infected and the prevalence, understand the detection rate of pathogens infected, and the prevalence trend, and then take scientific preventive measures. And compared with other detection kits currently available in the market, the detection limit of the kit provided in the present invention for Aspergillus and Candida can reach 500 copies / mL at the lowest, which greatly improves the sensitivity of detection.

[0041] (2) This kit is in the form of a fully premixed solution, that is, the primer probe, buffer and taq enzyme are premixed into one tube. When using, you only need to thaw, mix and dispense, which eliminates the step of preparing the reaction system and greatly improves work efficiency. In addition, the present invention optimizes the amplification procedure and the fluorescence acquisition time, greatly shortening the detection reaction time, and can shorten the detection time to within 1 hour.

[0042] (3) The kit provided in the present invention uses locked nucleic acid to modify the bases at specific sites on the primers, thereby improving the amplification efficiency of the primers and further improving the detection sensitivity while ensuring specificity. Compared with the primer sequence not modified with locked nucleic acid, the detection sensitivity of the present invention is improved by about 5 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a graph showing the detection results of human herpesvirus type 5, human herpesvirus type 6, human herpesvirus type 7 and human herpesvirus type 8 detected by an RT-PCR kit for simultaneously detecting multiple human herpesviruses in an embodiment of the present invention;

[0044] Figure 2 is a graph showing the experimental results of the detection limits of target sequences of various detection indicators using the primers and probes of the present invention in Experimental Example 1 of the present invention;

[0045] Figure 3 Comparison of the results of the locked nucleic acid modified primer system and the conventional primer system for the detection of human herpes virus type 5 samples in Experimental Example 2 of the present invention;

[0046] Figure 4 Comparison of the results of the locked nucleic acid modified primer system and the conventional primer system for the detection of human herpes virus type 6 samples in Experimental Example 2 of the present invention;

[0047] Figure 5 Comparison of the results of the locked nucleic acid modified primer system and the conventional primer system for the detection of human herpes virus type 7 samples in Experimental Example 2 of the present invention;

[0048] Figure 6 Comparison of the results of the locked nucleic acid modified primer system and the conventional primer system for the detection of human herpes virus type 8 samples in Experimental Example 2 of the present invention;

[0049] Figure 7 This is a diagram showing the results of cross-reaction detection of other human herpes virus pathogens using the primer-probe detection system of the present invention in Example 4 of the present invention. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0051] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0052] Example

[0053] This embodiment provides a RT-PCR kit for simultaneously detecting multiple human herpes viruses and a method for using the same, wherein the detection kit is prepared using a 20 μL amplification reaction system as an example, and the preparation scheme is shown in Table 1 below:

[0054] Table 1: Reagent preparation table

[0055]

[0056]

[0057] The RT-PCR premix is ​​a mixed solution of buffer solution and taq enzyme, purchased from Novozymes, product name: PureAmp U+Super Multiple Probe qPCR PreMix (Low DNA), item number: UQN221-C1. The corresponding sequences of the primer probe names in Table 1 are shown in Table 2 below:

[0058] Table 2: Primer probe sequences and positions of primer locked nucleic acid modifications of the present invention

[0059]

[0060]

[0061] The specific bases "A+", "C+" and "G+" in the primer sequences in the above table are bases modified with locked nucleic acid, wherein the primer sequences for detecting human herpes virus type 5 are shown in SEQ ID No.1 and SEQ ID No.2, and the detection probe sequence is shown in SEQ ID No.9, the 5' end of which is labeled with a FAM fluorescent group and the 3' end is labeled with a fluorescent quenching group MGB;

[0062] The primer sequences for detecting human herpesvirus type 6 are shown in SEQ ID No. 3 and SEQ ID No. 4, and the detection probe sequence is shown in SEQ ID No. 10, with the 5' end labeled with a VIC fluorescent group and the 3' end labeled with a fluorescent quenching group MGB;

[0063] The primer sequences for detecting human herpesvirus type 7 are shown in SEQ ID No.5 and SEQ ID No.6, and the detection probe sequences are shown in SEQ ID No.11 and SEQ ID No.12, with the 5' end labeled with a ROX fluorescent group and the 3' end labeled with a fluorescent quenching group MGB;

[0064] The primer sequences for detecting human herpesvirus type 8 are shown in SEQ ID No. 7 and SEQ ID No. 8, the detection probe sequence is shown in SEQ ID No. 13, the 5' end is labeled with a CY5 fluorescent group, and the 3' end is labeled with a fluorescent quenching group MGB.

[0065] This embodiment also provides a method for using the above detection kit, comprising the following steps:

[0066] Step 1: Extract sample nucleic acid,

[0067] Specifically, nucleic acids of positive samples of human herpesvirus HHV-5, human herpesvirus HHV-6, human herpesvirus HHV-7, and human herpesvirus HHV-8 were extracted for verification of the detection system, and the indicators of each positive sample were confirmed by sequencing.

[0068] Step 2: Thaw and mix the reagents and then dispense them directly into 96-well plates or eight-well tubes. Add the extracted sample template, negative control, and positive quality control products into different PCR reaction tubes and place them into fluorescent quantitative PCR instrument for fluorescent PCR detection.

[0069] Specifically, vortex the prepared reagents to mix evenly, centrifuge them momentarily, and directly dispense them into a 96-well plate or eight-well tube strip, with 18 μL dispensed into each well; bring the dispensed reagents to the sample loading room, add 2 μL of the sample nucleic acid, negative control, and positive quality control products extracted and prepared in advance into different PCR reaction tubes, cover the tubes with caps or seal them with film, centrifuge them momentarily, and bring them to the amplification room for on-machine testing.

[0070] Step 3: After the reaction in step 2 is completed, set the automatic baseline adjustment and analyze the test results according to the amplification curve and Ct value.

[0071] Specifically, the reaction conditions were set as 95°C for 2 min in the pre-denaturation phase, 95°C for 5 s, 56°C for 30 s in the amplification phase, and 40 cycles. After the settings were completed, the detection program was run. The criteria for interpreting the amplification curve and Ct value results were as follows:

[0072] Positive: "S"-shaped amplification curve appears, Ct value ≤ 35; Suspicious: "S"-shaped amplification curve appears, but Ct value > 35;

[0073] Negative: No Ct value; or the curve exceeds the threshold, but does not show an "S" shape. For suspicious results, the experiment should be repeated. If an "S"-shaped amplification curve appears in the repeated experiment, and the negative control is not contaminated, it can be judged as positive.

[0074] The experimental results are as attached Figure 1 As shown, from the results, it can be seen that the detection kit of the present invention can be used to simultaneously detect human herpes virus HHV-5, human herpes virus HHV-6, human herpes virus HHV-7, and human herpes virus HHV-8 sample nucleic acids, all of which can be accurately detected and have good linearity.

[0075] Experimental Example 1: Detection Limit Experiment

[0076] Experimental method: According to the detection kit and detection method provided in the embodiment of the present invention, the reference samples of human herpes virus HHV-5, human herpes virus HHV-6, human herpes virus HHV-7, and human herpes virus HHV-8 were detected. The reference samples are artificially synthesized and accurately quantified nucleic acid samples. After the samples are gradiently diluted, the concentrations of 5000 copies / mL, 2500 copies / mL, 1000 copies / mL, 500 copies / mL, and 250 copies / mL solutions are selected for on-machine detection to verify the minimum detection limit and linearity of the kit.

[0077] The experimental results are as attached Figure 2 It can be seen that the detection limit reference of each index detected by the primers and probes of the present invention shows good linearity of the detection system, and the minimum detection limit of each index of the present invention can reach 500 copies / mL, while the minimum detection limit of similar multiple detection kits on the market or similar multiple detection methods reported in the literature is claimed to be 1000 copies / mL. Detection using the primers and probes of the present invention has better sensitivity.

[0078] Experimental Example 2: Effect of Locked Nucleic Acid Modification on Detection Sensitivity

[0079] Experimental method: According to the detection kit and detection method provided in the embodiment of the present invention, conventional primers that have not been modified with locked nucleic acid are set as control group 1, and human herpes virus HHV-5, human herpes virus HHV-6, human herpes virus HHV-7, and human herpes virus HHV-8 are detected respectively. The sample is consistent with the detection kit in the embodiment. This experimental example detects the positive sample nucleic acid of each index with a sample concentration of 1000 copies / mL to verify whether the locked nucleic acid modification can effectively improve the detection sensitivity of the detection kit of the present invention.

[0080] The experimental results are shown in Table 3 below. It can be seen from the table that the locked nucleic acid modified primer system of the present invention can advance the Ct value of each index channel detection by 1-3 compared with the primer system of control group 1, reflecting that the detection sensitivity of the present invention is improved by about 5 times compared with control group 1. At the same time, the signal value of the present invention is also significantly improved. The signal value of the modified primer system of the present invention is distributed between about 1300-2100, and the signal value of the conventional primer system is between 800-1300.

[0081] Table 3: Comparison of the test results of the primer system of the present invention and the primer system without locked nucleic acid modification

[0082]

[0083] Experimental Example 3: Effect of probe sequence on amplification signal value

[0084] Experimental method: According to the detection kit and detection method provided in the embodiment of the present invention, the probe sequence used was replaced as the control group 2, and human herpes virus HHV-5, human herpes virus HHV-6, human herpes virus HHV-7, and human herpes virus HHV-8 were detected respectively to verify that the probe sequence used in the present invention has specificity. The specific probe sequence information of the control group 2 is shown in Table 4 below:

[0085] Table 4: Probe sequence list of control group 2

[0086]

[0087]

[0088] The embodiment and comparative example 2 were compared in an experiment. The reaction system was prepared as in the reference embodiment, and the template loading amount was 2 μL. The loading template was a target sequence solution of each index, with a concentration of 2500 copies / μL. The ABI 7500 fluorescent PCR instrument was used for on-machine detection, and the differences in detection Ct values ​​and signal values ​​between the embodiment and the control group 2 were compared. The experimental results are shown in Table 5 below:

[0089] Table 5: Test results of Example and Control Group 2

[0090]

[0091] Analysis of experimental results: It can be seen from Table 5 that the probe sequence selected in the present invention is superior to the control group 2 in terms of signal value. The high signal value can indirectly improve the sensitivity. At the same time, the amplification curve with a high signal value is more standardized, which makes it easier to interpret the results when detecting low-concentration samples. In addition, it has better specificity.

[0092] Example 4: Specificity test of the primer-probe detection system of the present invention

[0093] Experimental method: According to the detection kit and detection method provided in the embodiment of the present invention, four other pathogen cultures or samples with similar symptoms, the same infection site or similar species as the pathogenic infection of the reagent indicator are selected to test whether the detection kit provided by the present invention is specific to human herpes virus HHV-5, human herpes virus HHV-6, human herpes virus HHV-7, and human herpes virus HHV-8. These four indicators include: herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus and Epstein-Barr virus.

[0094] The experimental results are as attached Figure 7 As shown. Figure 7 It can be seen that the detection kit provided by the present invention has no cross reaction with the other four herpes viruses and has good specificity.

Claims

1. A RT-PCR kit for simultaneously detecting multiple human herpes viruses, characterized in that: The kit is a fully premixed reagent, comprising a buffer solution for amplification, a taq enzyme and a primer probe for detection. The human herpes viruses detected by the kit include human herpes virus type 5, human herpes virus type 6, human herpes virus type 7 and human herpes virus type 8.

2. A RT-PCR kit for simultaneously detecting multiple human herpes viruses as claimed in claim 1, characterized in that: The primers include Forward primer for detection of human herpesvirus type 5: 5'-GGCCGTTACTGTCTGCA+GGA-3', as shown in SEQ ID No.1, reverse amplification primer: 5'-GGCCTCGTAGTGAAAATTAATG+GT-3', as shown in SEQ ID No.2; Forward primer for detection of human herpesvirus type 6: 5'-AAAGACCTAAATTGCCGCTAC+CT-3', as shown in SEQ ID No.3, Reverse amplification primer: 5'-GCAAGCTCATGAACATCGTC+A-3', as shown in SEQ ID No.4; Forward primer for detection of human herpesvirus type 7: 5'-CCAATCCTTCCGAAACCG+AT-3', as shown in SEQ ID No.5, Reverse amplification primer: 5'-CGGAAGTCACTGGAGTAATGA+CAA-3', as shown in SEQ ID No.6; Forward primer for detection of human herpesvirus type 8: 5'-CTGCGACATATTTCCCTGATC+C-3', as shown in SEQ ID No.7, Reverse amplification primer: 5'-TGGATCATGACTACGCTCACC+AT-3', as shown in SEQ ID No.

8.

3. A RT-PCR kit for simultaneously detecting multiple human herpes viruses as claimed in claim 2, characterized in that: The probe comprises: Probes for human herpesvirus type 5: 5'-CCGTATTGGTGCGCGATCTGTTCAA-3', as shown in SEQ ID No. 9; Probes for human herpesvirus type 6: 5'-TTAGATGGTGGTGAGCTGGGATCGGT-3', as shown in SEQ ID No. 10; Probe for human herpesvirus 7: 5'-CTCGCAGATTGCTTGTTGGCCATG-3', As shown in SEQ ID No.11; 5'-TCGGTAAGATAAATAAGCAATCA-3', as shown in SEQ ID No.12; For human herpesvirus type 8 probe: 5'-TTCATGCGAGCCACCAG-3', such as SEQ ID Shown in No.

13.

4. The RT-PCR kit for simultaneously detecting multiple human herpes viruses according to claim 3, characterized in that: The human herpes virus type 5 detection probe sequence SEQ ID No.9 has a FAM fluorescent group labeled at the 5' end and a fluorescent quenching group MGB labeled at the 3' end; The human herpes virus type 6 detection probe sequence SEQ ID No. 10 has a VIC fluorescent group labeled at the 5' end and a fluorescent quenching group MGB labeled at the 3' end; The detection probe sequences of human herpes virus type 7, SEQ ID No. 11 and SEQ ID No. 12, are labeled with a ROX fluorescent group at the 5' end and a fluorescent quenching group MGB at the 3' end; The detection probe sequence of human herpes virus type 8, SEQ ID No. 13, has a CY5 fluorescent group labeled at the 5' end and a fluorescent quenching group MGB labeled at the 3' end.

5. A RT-PCR kit for simultaneously detecting multiple human herpes viruses as claimed in claim 2, characterized in that: The specific bases "A+", "C+" and "G+" in the primer sequence are locked nucleic acid modified bases.

6. The RT-PCR kit for simultaneously detecting multiple human herpes viruses according to claim 2, characterized in that: The final concentration of the primers in the reagent is 444 nM, and the final concentration of the probe is 222 nM.

7. A method for using a RT-PCR kit for simultaneously detecting multiple human herpes viruses as described in any one of claims 1 to 6, characterized in that: The steps include: Step 1: Extract sample nucleic acid; Step 2: Thaw and mix the reagents and then dispense them directly into 96-well plates or eight-well tubes. Add the extracted sample template, negative control, and positive quality control products into different PCR reaction tubes, cover the tubes, place them in the fluorescence quantitative PCR instrument, set the reaction conditions, and perform fluorescence PCR detection. Step 3: After the reaction in step 2 is completed, set the automatic baseline adjustment and analyze the test results according to the amplification curve and Ct value.

8. The method for using the RT-PCR kit for simultaneously detecting multiple human herpes viruses according to claim 7, characterized in that: The reaction conditions in step 2 are as follows: 95° C. for 2 min in the pre-denaturation stage; 95° C. for 5 s and 56° C. for 30 s in the amplification stage, and 40 cycles.

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