A dual real-time fluorescent RT-PCR detection primer, probe, kit and detection method for Hantan virus and Seoul virus

Abstract

The invention discloses a dual real-time fluorescent RT-PCR detection primer, probe and kit for Hantan virus and Seoul virus, and relates to the field of biotechnology application. The method has high sensitivity and strong specificity, and can realize rapid and accurate detection of Hantan virus and Seoul virus in clinical and host animal samples. The nucleotide sequences of the detection primer and the detection probe are as follows: F1: 5'-GACAACAAAGGGACTAGA-3'; R1: 5'-GTGCATTGGGTAGAGATA-3'; F2: 5'-TGGCTTCAAAAACTGTGG-3'; R2: 5'-GTCCAGTTGTATTCCCAT-3'; P1: 5'-ATTCGGTTCAAGGATGATAGC-3'; P2: 5'-TGATTGTGTGCGTCTGAGGT-3'.

Description

Technical Field

[0001] The invention relates to the field of biotechnology application, in particular to a double real-time fluorescent RT-PCR detection primer, a probe, a kit and a detection method for Hantaan virus and Seoul virus. Background Art

[0002] Hantavirus (HV) is an RNA virus of the genus Hantavirus in the family Bunyaviridae, which can cause hemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonary syndrome. The discovered HV can be divided into at least 40 serotypes or genotypes. There are many types of HV found in host animals in my country, among which Hantaan virus (HTNV) and Seoul virus (SEOV) are the most prevalent HVs in my country and are currently confirmed to cause HFRS in humans in my country. HFRS has an acute onset, rapid progression, and a high mortality rate.

[0003] Typical HFRS cases present with fever, shock, bleeding, and renal damage, or go through five stages, and diagnosis is not difficult; however, it is very difficult to make a clear diagnosis for patients with atypical clinical manifestations or in the early stages of fever.

[0004] Real-time fluorescence PCR is widely used in the early diagnosis of viral infectious diseases, but the current real-time fluorescence RT-PCR method for HV mostly detects a single type of HV, which cannot meet the needs of HFRS HTNV and SEOV mixed epidemic areas in most provinces and cities across the country. Therefore, the development of a fluorescent RT-PCR method that can detect both HTNV and SEOV Hantaviruses is of great significance for the early diagnosis of HFRS and epidemiological investigations. Summary of the invention

[0005] The object of the present invention is to provide a dual real-time fluorescent RT-PCR detection primer, a probe, a kit and a detection method for Hantaan virus and Seoul virus, which has high sensitivity and strong specificity and can realize rapid and accurate detection of Hantaan virus and Seoul virus in clinical and host animal samples.

[0006] To achieve the above object, the present invention provides the following technical solution: a dual real-time fluorescent RT-PCR detection primer and detection probe for Hantaan virus and Seoul virus, wherein the nucleotide sequences of the detection primer and detection probe are as follows:

[0007] F1: 5'-GACAACAAAGGGACTAGA-3';

[0008] R1: 5'-GTGCATTGGGTAGAGATA-3';

[0009] F2: 5'-TGGCTTCAAAAACTGTGG-3';

[0010] R2: 5'-GTCCAGTTGTATTCCCAT-3';

[0011] P1: 5'-ATTCGGTTCAAGGATGATAGC-3';

[0012] P2: 5'-TGATTGTGTGCGTCTGAGGT-3'.

[0013] Preferably, the fluorescent group labeled at the 5' end of the sequence in the detection probe includes one of FAM, HEX, VIC, CY5, and JOE;

[0014] The quenching group labeled at the 3' end of the sequence in the detection probe includes one of TAMRA, MGB, and BHQ;

[0015] The fluorescent groups labeled at the 5' ends of the detection probes P1 and P2 are different.

[0016] A dual real-time fluorescence RT-PCR detection kit for Hantaan virus and Seoul virus contains dual real-time fluorescence RT-PCR detection primers.

[0017] A dual real-time fluorescent RT-PCR detection kit for Hantaan virus and Seoul virus contains dual real-time fluorescent RT-PCR detection probes.

[0018] A dual real-time fluorescence RT-PCR detection method for Hantan virus and Seoul virus comprises the following steps:

[0019] S1: Extract viral RNA from samples;

[0020] S2: Using the extracted viral RNA as a template, fluorescent RT-PCR amplification was performed using primers F1, R1, F2 and R2 and probes P1 and P2;

[0021] S3: Determine the virus type in the sample based on the amplification curve.

[0022] Preferably, the fluorescent RT-PCR amplification reaction system in S2 is:

[0023] RT-PCR buffer 12.5 μL;

[0024] Enzyme Mix 1 μL;

[0025] 25 μM primer F1 0.5 μL;

[0026] 25 μM Primer R1 0.5 μL;

[0027] 25 μM primer F2 0.5 μL;

[0028] 25 μM Primer R2 0.5 μL;

[0029] 25 μM probe P1 0.5 μL;

[0030] 25 μM probe P2 0.5 μL;

[0031] ddH2O 3.5μL;

[0032] RNA template 5 μL;

[0033] Total volume 25 μL.

[0034] Preferably, the fluorescent RT-PCR amplification reaction conditions in S2 are:

[0035] 45℃ 10min;

[0036] 95℃ 10min;

[0037] 95℃ 15s;

[0038] 55℃ 45s (collect fluorescence).

[0039] Preferably, the fluorescent RT-PCR amplification reaction conditions include 45 cycles of amplification reaction at 95° C. for 15 s and amplification reaction at 55° C. for 45 s.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. In the invention, viral RNA is first extracted from the sample, and the extracted viral RNA is used as a template, and the primer pair F1: 5'-GACAACAAAGGGACTAGA-3' / R1: 5'-GTGCATTGGGTAGAGATA-3' and F2: 5'-TGGCTTCAAAAACTGTGG-3' / R2: 5'-GTCCAGTTGTATTCCCAT-3' and the probe P1: 5'-ATTCGGTTCAAGGATGATAGC-3' and P2: 5'-TGATTGTGTGCGTCTGAGGT-3' are used for fluorescent RT-PCR amplification, and the type of virus in the sample is determined according to the amplification curve. The method is simple to operate, and only one reaction is required to realize the detection of Hantan virus and Seoul virus. The entire operation process does not take 3 hours, and the analysis can be performed accurately, quickly and with high throughput, which is conducive to the promotion and application in clinical practice;

[0042] 2. The dual real-time fluorescent RT-PCR detection method for Hantaan virus and Seoul virus used in this invention has good specificity and high sensitivity. Using HTNV and SEOV recombinant plasmids as standards, it can detect a minimum of 10 copies / μL of recombinant positive plasmids, and can achieve rapid and accurate detection of Hantaan virus and Seoul virus in clinical and host animal samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is the fluorescence detection amplification curve diagram of the SEOV standard of the present invention;

[0044] Figure 2 It is the fluorescence detection amplification curve diagram of the HTNV standard of the present invention;

[0045] Figure 3 A specific detection diagram for the method of establishing the present invention;

[0046] Figure 4 This is a test chart of 10 serum samples of hemorrhagic fever with renal syndrome cases according to the present invention. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present invention will be described clearly and completely 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, rather than all the embodiments.

[0048] Example 1 Dual real-time fluorescence RT-PCR detection method and use of Hantaan virus and Seoul virus

[0049] 1-1 Primers and probes

[0050] After screening a large number of designed primers and probes, it was found that primer pairs F1 / R1, F2 / R2 and probes P1 and P2 had the best effect in detecting Hantaan virus and Seoul virus at the same time. Their base sequences are shown below:

[0051] F1: 5'-GACAACAAAGGGACTAGA-3';

[0052] R1: 5'-GTGCATTGGGTAGAGATA-3';

[0053] F2: 5'-TGGCTTCAAAAACTGTGG-3';

[0054] R2: 5'-GTCCAGTTGTATTCCCAT-3';

[0055] P1: 5'-ATTCGGTTCAAGGATGATAGC-3';

[0056] P2: 5'-TGATTGTGTGCGTCTGAGGT-3'.

[0057] The fluorescent group labeled at the 5' end of probe P1 is FAM, and the quencher group labeled at the 3' end is BHQ1;

[0058] The fluorescent group labeled at the 5' end of probe P2 is VIC, and the quenching group labeled at the 3' end is BHQ2.

[0059] 1-2 Viral nucleic acid extraction

[0060] The viral nucleic acid extraction reagent used was QIAamp Viral RNA Mini Kit (QIAGEN, extraction method and reagents were carried out according to the instructions).

[0061] 1-3 Use AgPath-ID TMOne-step RT-PCR Kit from Thermo Fisher Scientific (the commercial HTN / SEOV real-time fluorescence RT-PCR kit is relatively expensive, with the reagent cost of each sample being as high as RMB 100, which is difficult to adapt to China's national conditions and popularize and promote nationwide; the current price of AgPath-ID TMOne-step RT-PCR Kit from Thermo Fisher Scientific is about RMB 15 per person, and the cost of probes and primers for each sample is about RMB 5. Therefore, the reagent cost of this kit for each sample is only about RMB 20. It is roughly estimated that the reagent cost of this method is only about 1 / 5 of that of the commercial fluorescence RT-PCR kit. The detection cost is low, which can greatly save the detection cost and is very suitable for large-scale detection).

[0062] Add the reaction premix (RT-PCR buffer) and enzyme (Enzyme Mix) to the PCR reaction tube, and then add the probe and primer. The specific reaction system is:

[0063] RT-PCR buffer 12.5 μL;

[0064] Enzyme Mix 1 μL;

[0065] 25 μM primer F1 0.5 μL;

[0066] 25 μM Primer R1 0.5 μL;

[0067] 25 μM primer F2 0.5 μL;

[0068] 25 μM Primer R 2 0.5 μL;

[0069] 25 μM probe P1 0.5 μL;

[0070] 25 μM probe P2 0.5 μL;

[0071] ddH2O 3.5μL;

[0072] RNA template 5 μL;

[0073] Total volume 25 μL.

[0074] 1-4 Fluorescence RT-PCR amplification reaction conditions are:

[0075] 45℃ 10min;

[0076] 95℃ 10min;

[0077] 95℃ 15s;

[0078] 55°C 45s (collecting fluorescence);

[0079] 95°C for 15 s and 55°C for 45 s were cycled 45 times.

[0080] 1-5 Quality Control Standards

[0081] The negative control had no Ct value and no amplification curve;

[0082] When the Ct value of the positive control is ≤30.0 and a typical amplification curve appears.

[0083] 1-6 Result Expression and Judgment

[0084] Samples with a Ct value of ≤35.0 were judged as positive, indicating the presence of Hantaan virus / Seoul virus in the sample;

[0085] Samples without Ct values ​​and amplification curves were judged as negative, indicating that Hantaan virus / Seoul virus was absent in the samples;

[0086] Samples with Ct values ​​between 35 and 40 must be redone. If there is no Ct value in the redone results, it is negative, otherwise it is positive.

[0087] 1-7 Time advantage

[0088] Nucleic acid extraction takes about 40 minutes (automatic nucleic acid extractor only needs 30 minutes), and one-step real-time fluorescence RT-PCR takes about 100 minutes. Therefore, the time from receiving the sample to obtaining the test results is only 2.5 to 3 hours, which greatly saves detection time.

[0089] Example 2 Sensitivity Experiment

[0090] 2-1 Positive standard: Clone the SEOV and HTNV S genes (target fragment length 1311 bp) into the pGEM ® -T (Promega) vector to prepare the recombinant plasmid as a positive control.

[0091] 2-2 Purify the positive plasmids of SEOV and HTNV, measure the concentration by BioDrop ultra-micro protein nucleic acid analyzer UV spectrophotometer, and then calculate the copy number according to Avogadro constant, and dilute the positive plasmids to 1.0×10 7 copies / μL, 1.0×10 6 copies / μL, 1.0×10 5 copies / μL, 1.0×10 4 copies / μL, 1.0×10 3 copies / μL, 1.0×10 2 copies / μL, 1.0×10 1 copies / μL, 1.0×10 0 copies / μL, 1.0×10 - 1 copies / μL, the gradient diluted positive standard was tested according to Example 1 to verify the sensitivity of the established method,

[0092] The results are as follows Figure 1 and Figure 2 shown.

[0093] 2-3 Conclusion

[0094] Depend on Figure 1 and Figure 2 It can be seen that the detection method of the present invention shows a significant decrease in fluorescence signal as the nucleic acid concentration decreases. 7 -10 -1 ), 7 of them showed amplification curves (10 7 The lowest detectable concentration of HTNV and SEOV was 10 copies / μL.

[0095] Example 3 Specificity Experiment

[0096] 3-1 Using influenza A virus, dengue virus, new Bunyavirus, Zika virus, and new coronavirus positive nucleic acid stored in this laboratory as templates, and setting up positive controls (positive plasmids) and negative controls, the method of Example 1 was used for detection to verify the specificity of the established method. The results are as follows Figure 3 shown.

[0097] 3-2 Conclusion

[0098] Depend on Figure 3 It can be seen that the positive control has an amplification curve, while the other 5 viral nucleic acid samples and the negative control have no amplification curve, indicating that the detection method has good specificity.

[0099] Example 4 Detection of serum samples from hemorrhagic fever with renal syndrome cases

[0100] 4-1 Ten serum samples of hemorrhagic fever with renal syndrome cases collected in 2017 and frozen at -70°C were selected, and dual real-time fluorescence RT-PCR detection of Hantaan virus and Seoul virus was performed according to the method in Example 1, and positive and negative controls were set at the same time.

[0101] 4-2 Conclusion

[0102] Results Figure 4 , among which the negative control showed no amplification curve, and the positive control showed an amplification curve. Among the 10 serum samples of hemorrhagic fever with renal syndrome, 9 were HTNV type (9 HTNV virus gene sequences were accessed in GenBank as: MT782310-MT782318), and 1 was SEOV type (no gene sequence was obtained).

[0103] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A dual real-time fluorescent RT-PCR detection primer and detection probe for Hantaan virus and Seoul virus, characterized in that: The nucleotide sequences of the detection primers and detection probes are as follows: F1: 5'-GACAACAAAGGGACTAGA-3'; R1: 5'-GTGCATTGGGTAGAGATA-3'; F2: 5'-TGGCTTCAAAAACTGTGG-3'; R2: 5'-GTCCAGTTGTATTCCCAT-3'; P1: 5'-ATTCGGTTCAAGGATGATAGC-3'; P2: 5'-TGATTGTGTGCGTCTGAGGT-3'; The fluorescent group labeled at the 5' end of the sequence in the detection probe includes one of FAM, HEX, VIC, CY5, and JOE; The quenching group labeled at the 3' end of the sequence in the detection probe includes one of TAMRA, MGB, and BHQ; The fluorescent groups labeled at the 5' ends of the detection probes P1 and P2 are different.

2. A dual real-time fluorescent RT-PCR detection kit for Hantaan virus and Seoul virus, based on a dual real-time fluorescent RT-PCR detection primer and detection probe for Hantaan virus and Seoul virus in claim 1, characterized in that: The detection kit contains dual real-time fluorescent RT-PCR detection primers.

3. A dual real-time fluorescent RT-PCR detection kit for Hantaan virus and Seoul virus, based on a dual real-time fluorescent RT-PCR detection primer and detection probe for Hantaan virus and Seoul virus in claim 1, characterized in that: The detection kit contains dual real-time fluorescent RT-PCR detection probes.

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