Primer combination for detecting Nipah virus, Rift Valley fever virus and Japanese encephalitis virus and application thereof

Abstract

The invention discloses a primer combination for detecting Nipah virus, Rift Valley fever virus and Japanese encephalitis virus and application of the primer combination, belongs to the technical field of molecular biology, and aims to overcome the technical defects in the prior art. Specific primers and probes are designed for targeting a Nipah virus N gene, a Rift Valley fever virus L gene and a Japanese encephalitis virus 5 'UTR, nucleotide sequences are as shown in SEQ ID NO: 1-SEQ ID NO: 12, the primer combination can detect 200 copies / mL of virus RNA with high specificity and high sensitivity, meanwhile, differential diagnosis of three pathogens is achieved in a single reaction system, and the primer combination has the advantages of high specificity and high sensitivity. The technical support is provided for early screening of diseases and prevention and control of epidemic situations, and the domestic technical short board is filled up.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a primer combination for detecting Nipah virus, Rift Valley fever virus and Japanese encephalitis virus and an application thereof. Background Art

[0002] Nipah virus (NiV), Rift Valley fever virus (RVFV) and Japanese encephalitis virus (JEV) are all zoonotic pathogens. Nipah virus is transmitted through a fruit bat-livestock-human chain, with a mortality rate of 40%-75%. Rift Valley fever virus is transmitted by mosquitoes or contact with infected animals, and can cause hemorrhagic fever syndrome. Therefore, RVFV is a typical high-risk pathogen that can cause new and emerging infectious disease outbreaks. Japanese encephalitis virus, as the main pathogen of viral encephalitis in Asia, causes about 68,000 cases of infection each year, of which 20%-30% leave neurological sequelae. The clinical manifestations of the three are highly overlapping in the acute phase, all accompanied by fever, headache, neurological symptoms, etc.

[0003] Existing detection methods for Nipah virus (NiV), Rift Valley fever virus (RVFV) and Japanese encephalitis virus (JEV) include: serological testing, virus isolation and culture, and molecular biology detection technology. Serological testing is susceptible to cross-reaction interference; traditional virus isolation methods require BSL-3 / BSL-4 level laboratories and related equipment, and the culture cycle is long, taking more than 7 days; and although single pathogen nucleic acid detection can provide good detection performance, it is not sensitive enough to variants and the multi-pathogen separation process is cumbersome and costly, which seriously restricts the efficiency of early differential diagnosis and precise prevention and control of the epidemic. Therefore, it is urgent to develop a multiple detection scheme with broad spectrum, high sensitivity and grassroots adaptability to meet the challenges of regional mixed infections and provide key technical support for improving the emergency response capabilities of sudden outbreaks. Summary of the invention

[0004] In order to solve the technical bottlenecks existing in the existing detection methods for multiple detection of Nipah virus (NiV), Rift Valley fever virus (RVFV) and Japanese encephalitis virus (JEV), the present invention provides a primer combination for detecting Nipah virus, Rift Valley fever virus and Japanese encephalitis virus, the primer combination comprising:

[0005] Specific primers and probes designed to target the Nipah virus N gene, the nucleotide sequences of the specific primers are shown in SEQ ID NO: 1-SEQ ID NO: 2, and the nucleotide sequence of the probe is shown in SEQ ID NO: 3;

[0006] Specific primers and probes designed to target the Rift Valley fever virus L segment polymerase gene, the nucleotide sequences of the specific primers are shown in SEQ ID NO:4-SEQ ID NO:5, and the nucleotide sequence of the probe is shown in SEQ ID NO:6;

[0007] Specific primers and probes designed to target the 5'UTR of Japanese encephalitis virus, the nucleotide sequences of the specific primers are shown in SEQ ID NO:7-SEQ ID NO:8, and the nucleotide sequence of the probe is shown in SEQ ID NO:9;

[0008] The primer combination of the present invention also includes an upstream primer, a downstream primer, and a probe of an internal reference gene β-actin, and the nucleotide sequences are shown in SEQ ID NO: 10-SEQ ID NO: 12.

[0009] The present invention uses a multiplex qPCR method to detect different targets to achieve simultaneous detection of Nipah virus, Rift Valley fever virus and Japanese encephalitis virus in a single reaction tube, making up for the defects of existing single pathogen nucleic acid detection and providing a targeted strategy for subsequent treatment and disease prevention. The primer combination of the present invention has high detection sensitivity, which can reach 200 copies / mL, strong specificity, and more accurate detection.

[0010] The fluorescent groups of the probes in the composition of the present invention are different from each other and do not interfere with each other.

[0011] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, FAM, HEX, ROX and CY5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other.

[0012] In the present invention, the fluorescent reporter group of the probe for detecting Nipah virus is FAM; the fluorescent reporter group of the probe for detecting Rift Valley fever virus is ROX; the fluorescent reporter group of the probe for detecting Japanese encephalitis virus is HEX, and the fluorescent reporter group of the probe for detecting internal standard β-actin is CY5; the 3' end of the probe also has a quenching group, such as BHQ1 or BHQ2, BHQ3.

[0013] Another object of the present invention is to use the above-mentioned primer combination in the preparation of a detection reagent or kit for simultaneously detecting Nipah virus, Rift Valley fever virus and Japanese encephalitis virus, wherein the detection reagent or kit also includes other conventional reagents for multiplex fluorescence PCR, such as PCR reaction buffer, RT-PCR enzyme mixture, negative quality control product, and positive quality control product.

[0014] The negative control product is DEPC H 2 O, at least one of normal saline; the positive quality control product is at least one of Nipah virus, Rift Valley fever virus, Japanese encephalitis virus and β-actin fragment nucleic acid, pseudovirus, etc.

[0015] The method of using the above-mentioned multiplex fluorescence quantitative PCR detection reagent is as follows:

[0016] (1) Extracting or releasing nucleic acid from the sample to be tested;

[0017] (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the primer combination;

[0018] (3) Obtain and analyze the results.

[0019] In the present invention, the sample used for detection can be serum, oropharyngeal or nasopharyngeal secretions, cerebrospinal fluid, etc., but is not limited thereto.

[0020] The reaction conditions of the fluorescent quantitative PCR are:

[0021] The first stage: reverse transcription, the reaction conditions are: temperature 42℃, time 5min;

[0022] The second stage: pre-amplification, the reaction conditions are: pre-denaturation, temperature is 95℃, time is 30s, 1 cycle; denaturation, temperature is 95℃, time is 5s; annealing, temperature is 55℃~60℃, decrease 1℃ each cycle, time is 30s; extension, temperature is 72℃, time is 30s, 5 cycles;

[0023] The third stage: amplification and detection, the reaction conditions are: denaturation, temperature is 95 ° C, time is 5s; annealing, temperature is 58 ° C, time is 30s, 40 cycles, fluorescence is collected during the annealing stage.

[0024] Advantages and technical effects of the present invention:

[0025] (1) Based on the multiplex fluorescent PCR technology, the present invention develops a primer combination for detecting Nipah virus, Rift Valley fever virus and Japanese encephalitis virus. The combination has the advantages of being convenient and rapid, and can simultaneously realize triple pathogen detection, overcoming the cumbersomeness of the single pathogen step-by-step detection process and improving the detection efficiency;

[0026] (2) The method of the present invention has the advantages of high sensitivity, strong specificity and high accuracy, and the minimum detection limit can reach 200 copies / mL. In addition, positive quality control is set to participate in the quality monitoring of the entire process such as nucleic acid extraction and PCR amplification to avoid the generation of false positive or false negative results and improve the detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The test results of the nucleic acid detection kit samples for Nipah virus, Rift Valley fever virus, and Japanese encephalitis virus;

[0028] Figure 2 Specificity verification results for the nucleic acid detection kits for Nipah virus, Rift Valley fever virus, and Japanese encephalitis virus;

[0029] Figure 3 This is the result of the Nipah virus sensitivity test;

[0030] Figure 4 The sensitivity test results for Rift Valley fever virus;

[0031] Figure 5 This is the sensitivity test result of Japanese encephalitis virus. DETAILED DESCRIPTION

[0032] The technical scheme of the present invention is further described in detail below through examples, but the content of the present invention is not limited thereto. The methods in the present examples are conventional methods unless otherwise specified, and the materials and reagents used are obtained from commercial channels or prepared according to conventional methods unless otherwise specified;

[0033] Example 1: Design of primer probe and synthesis of positive quality control

[0034] 1. Design of specific primers and probes for Nipah virus, Rift Valley fever virus and Japanese encephalitis virus

[0035] The viral reference sequences of Nipah virus, Rift Valley fever virus and Japanese encephalitis virus were downloaded from NCBI, with sequence IDs: MK575070.1, MT890713.1, MK673582.1, KX944858.1, MG659830.1, MG659846.1, KX779522.1, KT957422.1, KY650726.1. Then, the MAFFT v7.037b program was used for sequence alignment. After a systematic evaluation of the conservation and specificity of the gene sequences, we designed specific primers and probes targeting the Nipah virus N gene, the Rift Valley fever virus L gene, and the Japanese encephalitis virus 5'UTR, respectively. At the same time, primers and probes were designed for the internal reference gene β-actin for the full-process detection of the monitoring method to avoid false positive or false negative results. The primer and probe sequences are shown in the following table:

[0036] Table 1 Primer probe sequence information used in the present invention

[0037]

[0038]

[0039] 2. Positive quality control synthesis

[0040] In this example, Nipah virus, Rift Valley fever virus, Japanese encephalitis virus and β-actin gene sequences were obtained by chemical synthesis and cloned into pET28b-MS2 vector, and armor RNA was prepared, concentrated and purified in Escherichia coli. The armor RNA obtained is the RNA nucleic acid sequence of the capsid protein of MS2 bacteriophage, which can be used as an internal reference product for viral RNA nucleic acid extraction experiments and qPCR detection experiments. The preparation steps are as follows:

[0041] (1) The Nipah virus N gene, the Rift Valley fever virus L gene, the Japanese encephalitis virus 5'-UTR and the β-actin detection target gene sequence are tandemly synthesized into a target gene (sequence shown in SEQ ID NO: 13), and inserted into the pET-28b-MS2 vector (prepared by referring to the method in the invention application CN 108795961 A). The pET-28b-MS2 phage vector contains the 5' non-coding region sequence, the mature enzyme protein gene sequence, the capsid protein gene sequence, the packaging site and the replicase gene sequence of the phage MS2, and can autonomously perform pseudovirus packaging;

[0042] (2) The successfully constructed pET-28b-MS2 expression plasmid containing the Nipah virus N gene, the Rift Valley fever virus L gene, the Japanese encephalitis virus 5'-UTR and the β-actin detection target gene was transferred into the Escherichia coli prokaryotic expression strain BL21, and 0.5mM IPTG was used to induce expression for 8 hours. After ultrasonic disruption, centrifugation and collection of the precipitate were performed to obtain armor RNA virus-like particles containing the Nipah virus N gene, the Rift Valley fever virus L gene, the Japanese encephalitis virus 5'-UTR and the β-actin detection target gene. The construction of the armor RNA virus-like particles was entrusted to Anhui General Biotechnology;

[0043] Virus-like particle RNA was extracted and the concentration was measured by UV spectrophotometer. The copy number of RNA was calculated according to the detected target sequence and concentration. The calculation formula is as follows:

[0044]

[0045] Example 2: Establishment of multiplex RT-qPCR reaction system

[0046] In this example, the one-step RT-qPCR kit (Cat. No.: AG11708) of Aikerui Biotechnology Co., Ltd. was used to prepare the multiplex qPCR reaction system according to the instructions of the kit. The primers and probes used were as described in Example 1. The template was a pseudovirus RNA containing the Nipah virus N gene, the Rift Valley fever virus L gene, the Japanese encephalitis virus 5'-UTR, and the β-actin detection target gene. The concentration was 5×10 3 copies / mL;

[0047] The multiplex qPCR reaction system is as follows:

[0048]

[0049] The reaction procedure is as follows:

[0050]

[0051] By optimizing the annealing temperature of the multiplex qPCR reaction, set the gradient annealing temperature to 52°C, 54°C, 56°C, 58°C, 60°C, 62°C, and 64°C. Establish the optimal ratio of the reaction system components of the multiplex RT-qPCR by adjusting the primer concentration (0.1μM to 1μM) and the probe concentration (0.1μM to 0.5μM).

[0052] Table 2 Ct values ​​at different annealing temperatures

[0053]

[0054]

[0055] Table 3 Ct values ​​at different primer-probe concentration ratios

[0056]

[0057] The optimal conditions were selected according to the CT value and fluorescence intensity of each detection channel. In Table 2, the CT value increased with the increase of annealing temperature, but there was no significant difference. When the annealing temperature was 58°C, a better CT value could be obtained, so 58°C was selected as the optimal primer annealing temperature. In Table 3, when the primer concentration was 0.1 μmol / L, no matter how much or how little the probe concentration was, it had a greater impact on the amplification efficiency, and the CT value was too large. When the primer concentration was 0.5 μmol / L and 1 μmol / L, better detection results could be obtained. Considering the detection CT value, fluorescence intensity, detection cost, etc., the final preferred primer concentration was 0.5 μmol / L and the probe concentration was 0.25 μmol / L.

[0058] Example 3: Preparation and use of a nucleic acid detection kit for Nipah virus, Rift Valley fever virus, and Japanese encephalitis virus

[0059] Using the primer combination designed in Example 1 and the optimal primer-probe concentration ratio prioritized in Example 2, a nucleic acid detection kit for Nipah virus, Rift Valley fever virus, and Japanese encephalitis virus was prepared. The kit included a primer combination, a PCR reaction buffer, an RT-qPCR enzyme mixture, a negative quality control product, and a positive quality control product. Taking 48 tests / box as an example, the components of the kit are as follows;

[0060]

[0061]

[0062] The method of using the Nipah virus, Rift Valley fever virus, and Japanese encephalitis virus nucleic acid detection kit is as follows:

[0063] (1) Nucleic acid extraction

[0064] Take 200 μL of serum, oropharyngeal or nasopharyngeal secretions, and cerebrospinal fluid, and use a viral DNA / RNA nucleic acid extraction kit to obtain viral RNA;

[0065] (2) Preparation of detection reaction system

[0066] The kit of the present invention can realize triple pathogen detection of Nipah virus, Rift Valley fever virus and Japanese encephalitis virus in a single reaction tube, and the reaction system is prepared as follows:

[0067] Reagent components Volume per reaction (μL) PCR reaction buffer 20μL RT-qPCR Enzyme Mix 1.6μL Primer probe combination 12μL <![CDATA[ddH 2 The]]> 1.4μL template 5μL Overall system 40μL

[0068] Note: The primer-probe combination is prepared according to the optimal concentration selected in Example 2.

[0069] (3) Use Shanghai Hongshi SLAN96-P fluorescence quantitative PCR instrument and set the qPCR reaction program as follows:

[0070]

[0071]

[0072] (4) Result analysis and judgment

[0073]

[0074] Note: CY5 channel is the internal reference channel. The internal reference test results must meet the following conditions: (1) If all pathogen detection targets are negative, the internal reference (CY5) channel must meet the CT value ≤ 38; (2) If any one or more pathogen detection results are positive, the CT value of the internal reference (CY5) channel is not limited; (3) If any reaction does not meet the above two conditions, it is judged that the internal reference test is unqualified and the sample needs to be retested.

[0075] Example 4: Detection results of the primer combination test sample of the present invention

[0076] The primers and probes shown in Example 1 were used according to the method of Example 2 to extract nucleic acid from armored RNA virus-like particles containing the Nipah virus N gene, the Rift Valley fever virus L gene, the Japanese encephalitis virus 5'-UTR, and the β-actin detection target gene to obtain RNA nucleic acid for verification;

[0077] The test results showed that all fluorescent channels were able to detect specific amplification curves, indicating that the primer combination of the present invention can simultaneously perform differential diagnosis on Nipah virus, Rift Valley fever virus, and Japanese encephalitis virus. Figure 1 shown.

[0078] Example 5: Specificity and anti-interference test of the primer combination of the present invention

[0079] (1) Pathogen cross-reaction test

[0080] In this example, five pathogen samples with homology in nucleic acid sequences and prone to cause the same or similar clinical symptoms were selected as cross-reaction evaluation samples, and the detection method described in Example 3 was used to perform a cross-reaction test to evaluate the specificity of the composition of the present invention; the test results are shown in FIG. Figure 2 As shown in Table 4:

[0081] Table 4 Specific results of nucleic acid detection kits for Nipah virus, Rift Valley fever virus and Japanese encephalitis virus

[0082]

[0083]

[0084] (2) Anti-interference test of the primer combination of the present invention

[0085] In order to investigate the effect of endogenous / exogenous interfering substances on the performance of the detection results, the additives were diluted to the test concentration using virus preservation solution (Cat. No.: VTM-a-01, Xi'an Tianlong), and 500 copies / mL of pseudovirus particles (containing Nipah virus N gene, Rift Valley fever virus L gene, Japanese encephalitis virus 5'-UTR and β-actin detection target gene) from step (2) of Example 1 were mixed in; nucleic acid RNA was extracted, and the detection method in Example 3 was used to test common interfering substances;

[0086] The experimental results show that potential PCR inhibitors / interfering substances such as dexamethasone (50μg / mL), ganciclovir (100μg / mL), zanamivir (100μg / mL), ribavirin (100μg / mL), clindamycin (50μg / mL), tobramycin (50μg / mL), heme (10μg / mL), and hemoglobin (10%) have no significant effect on this kit. The amplification results of the PCR reaction solution in the presence of infectious substances are shown in the table below;

[0087]

[0088] Example 6: Sensitivity test of the primer combination of the present invention

[0089] In this example, the serum sample was collected using virus preservation solution (Cat. No.: VTM-a-01, Xi'an Tianlong) and mixed with 1×10 7 copies / mL of pseudovirion particles (prepared in Example 2), and gradiently diluted them to 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 500 and 200 copies / mL of templates were used to simulate clinical samples for sensitivity testing. Figure 3-Figure 5 As shown, the primer combination of the present invention has a good amplification efficiency for Nipah virus, Rift Valley fever virus, and Japanese encephalitis virus; when the input template amount is 200 copies / mL, 100% detection can be achieved, so the detection sensitivity of the composition of the present invention is set to 200 copies / mL.

[0090] In summary, the present invention is based on multiplex fluorescence quantitative PCR technology, targeted design of specific primer probes, and rapid differential diagnosis of three pathogens in a single reaction tube. This has great value in terms of clinical accurate and rapid diagnosis, treatment and patient prognosis of Nipah virus, Rift Valley fever virus and Japanese encephalitis virus, greatly improves detection efficiency, and is a diagnostic technology with good prospects.

Claims

1. A primer combination for detecting Nipah virus, Rift Valley fever virus and Japanese encephalitis virus, characterized in that: It includes specific primers and probes targeting Nipah virus, and the nucleotide sequences are shown in SEQ ID NO: 1-SEQ ID NO: 3; Specific primers and probes targeting Rift Valley fever virus, the nucleotide sequences of which are shown in SEQ ID NO:4-SEQ ID NO:6; Specific primers and probes targeting Japanese encephalitis virus, the nucleotide sequences of which are shown in SEQ ID NO:7-SEQ ID NO:

9.

2. The primer combination for detecting Nipah virus, Rift Valley fever virus and Japanese encephalitis virus according to claim 1, characterized in that: Also included are specific primers and probes for the internal reference gene β-actin, and the nucleotide sequences are shown in SEQ ID NO: 10-SEQ ID NO:

12.

3. Use of the primer combination for detecting Nipah virus, Rift Valley fever virus and Japanese encephalitis virus according to claim 2 in the preparation of a detection reagent or kit for simultaneously detecting Nipah virus, Rift Valley fever virus and Japanese encephalitis virus nucleic acids.

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