Primer group and kit for detecting Langhenipah virus and verification method of Langhenipah virus

Abstract

The invention discloses a primer group, a kit and a verification method for detecting a Langheng nipah virus, the primer group comprises a primer group A for detecting the Langheng nipah virus and a primer group B for detecting the Langheng nipah virus, the primer group A comprises at least one of a primer group N, a primer group M and a primer group P / V / W / C; the primer group B comprises at least one of a primer group sg-N, a primer group sg-M, a primer group sg-L, a primer group sg-P / V / W / C-1 and a primer group sg-P / V / W / C-2. Primers are designed based on a plurality of conserved regions (including an N region, an M region and a P / V / W / C region) in a Langheng nipah virus genome, multi-target detection is realized, false negative caused by mutation of a single target is effectively avoided, and the sensitivity is improved.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological detection, and in particular to a primer group, a kit and a verification method for detecting Langya henipavirus. BACKGROUND

[0002] Langya henipavirus is a newly discovered zoonotic paramyxovirus in recent years, belonging to the henipavirus genus. Phylogenetic analysis shows that Langya henipavirus is closely related to Mozang virus and nipah virus, with a genome homology of 68-75%. Unlike Hendra virus and nipah virus, which mainly use bats as their main host, Langya henipavirus is mainly detected in shrews, which expands the host range of henipavirus.

[0003] Members of the henipavirus genus can usually cause severe respiratory disease and encephalitis, with a mortality rate of 40-70%, and have been proven to have the ability to transmit from animals to humans and limited interpersonal transmission. Although the number of human infections with Langya henipavirus is currently limited, its pathogenesis, transmission route and epidemic potential are still unclear. However, in view of the lessons of previous pandemics, it is particularly important to develop molecular diagnostic techniques.

[0004] CN117737309A provides a quadruple qRT-PCR method for Langya henipavirus, Mozang virus, nipah virus and cedrovirus, which has good sensitivity, specificity and repeatability. However, this method is mainly aimed at bat swabs and simulated samples using plasmid DNA, and when faced with samples (such as saliva, nasopharyngeal swabs, sputum, blood, etc.) containing a large number of substances that may inhibit PCR reactions, the sensitivity will be reduced. SUMMARY

[0005] In order to improve the sensitivity of Langya henipavirus detection, the application provides a primer group, a kit and a verification method for detecting Langya henipavirus.

[0006] In a first aspect, the application provides a primer group A for detecting Langya henipavirus, which adopts the following technical solution: A primer group A for detecting Langya henipavirus, comprising at least one of primer group N, primer group M, primer group P / V / W / C; The primer set N comprises an upstream primer N-F and a downstream primer N-R, the sequence list of which is shown in SEQ ID NO. 1 and SEQ ID NO. 2 respectively; the primer set M comprises an upstream primer M-F and a downstream primer M-R, the sequence list of which is shown in SEQ ID NO. 5 and SEQ ID NO. 6 respectively; and the primer set P / V / W / C comprises an upstream primer P / V / W / C-F and a downstream primer P / V / W / C-R, the sequence list of which is shown in SEQ ID NO. 17 and SEQ ID NO. 18 respectively.

[0007] By adopting the technical solutions described above, the present application designs primers based on multiple conserved regions (including N region, M region and P / V / W / C region) in the genome of Langya Henipavirus, realizes multi-target detection, effectively avoids false negatives caused by mutations in a single target, and improves sensitivity. In addition, all primer sets have good amplification efficiency and linear correlation at an optimized melting temperature (about 56℃) and GC content (47.8%), and can be applied to a standard detection platform. Among them, the P / V / W / C region can express four proteins, namely phosphoprotein, V protein, W protein and C protein; the present application designs a pair of primers (primer set P / V / W / C) which can target the entire P / V / W / C region, realizes the detection of multiple overlapping gene fragments, and thus reduces the risk of missed detection.

[0008] In a second aspect, the present application provides a primer set B for detecting Langya Henipavirus, which adopts the following technical solutions: The primer set B for detecting Langya Henipavirus comprises at least one of primer set sg-N, primer set sg-M, primer set sg-L, primer set sg-P / V / W / C-1 and primer set sg-P / V / W / C-2. The primer set sg-N includes an upstream primer sg-N-F and a downstream primer sg-N-R, the sequence lists of which are shown in SEQ ID NO. 3 and SEQ ID NO. 4 respectively; the primer set sg-M includes an upstream primer sg-M-F and a downstream primer sg-M-R, the sequence lists of which are shown in SEQ ID NO. 7 and SEQ ID NO. 8 respectively; the primer set sg-L includes an upstream primer sg-L-F and a downstream primer sg-L-R, the sequence lists of which are shown in SEQ ID NO. 25 and SEQ ID NO. 26 respectively; the primer set sg-P / V / W / C-1 includes an upstream primer sg-P / V / W / C-1-F and a downstream primer sg-P / V / W / C-1-R, the sequence lists of which are shown in SEQ ID NO. 19 and SEQ ID NO. 20 respectively; and the primer set sg-P / V / W / C-2 includes an upstream primer sg-P / V / W / C-2-F and a downstream primer sg-P / V / W / C-2-R, the sequence lists of which are shown in SEQ ID NO. 21 and SEQ ID NO. 22 respectively.

[0009] By adopting the technical solution, the application realizes multi-target detection by designing primers for multiple conserved regions (including N region, M region, L region and P / V / W / C region) in the Langya Henipavirus genome, effectively avoids false negatives caused by mutations in a single target, and improves sensitivity. In addition, all primer sets have good amplification efficiency and linear correlation at an optimized melting temperature (about 60℃) and GC content (about 56%), and can be applied to high-specificity detection. Among them, the P / V / W / C region can express four proteins, namely phosphoprotein, V protein, W protein and C protein. The application designs two pairs of primers (primer set sg-P / V / W / C-1 and primer set sg-P / V / W / C-2) for the P / V / W / C region, which can cover different gene fragments in the region, ensuring effective detection when the virus mutates and improving the robustness of detection.

[0010] In a third aspect, the application provides a kit for detecting Langya Henipavirus, which adopts the following technical solution: A kit for detecting Langya Henipavirus includes primer set A for detecting Langya Henipavirus or primer set B for detecting Langya Henipavirus.

[0011] By adopting the technical solution, the application designs a kit for primer set A or primer set B, which can ensure high-specificity and high-sensitivity detection of multiple conserved gene regions of Langya Henipavirus. This not only significantly reduces the risk of missed detection caused by local variation of the virus genome, but also provides convenience for reliable detection in different application scenarios (such as preliminary screening and review, different instrument platforms).

[0012] In a fourth aspect, the application provides a method for verifying a primer set for detecting Langya henipavirus, which employs the following technical solution: A method for verifying a primer set for detecting Langya henipavirus, comprising the following steps: S1, obtaining a Langya henipavirus genome, cloning the Langya henipavirus genome into a pUC57 plasmid vector to obtain a recombinant plasmid; S2, transforming the recombinant plasmid into Escherichia coli Top10 cells, culturing in a culture medium containing ampicillin, then extracting plasmid DNA, purifying to obtain a purified recombinant plasmid; S3, performing fluorescent quantitative PCR on the purified recombinant plasmid using primer set A for detecting Langya henipavirus or primer set B for detecting Langya henipavirus, and determining whether the sample contains Langya henipavirus according to the fluorescent amplification signal of real-time fluorescent quantitative PCR.

[0013] By employing the above technical solution, the application provides a method for verifying a primer set for detecting Langya henipavirus. The method first prepares a high-purity and high-concentration recombinant plasmid by cloning the viral genome into a pUC57 vector and amplifying it in Escherichia coli, which is used as a standard for fluorescent quantitative PCR. On this basis, fluorescent quantitative PCR is performed using specific primer sets, and whether the sample contains Langya henipavirus can be determined by analyzing the fluorescent amplification signal. The standard prepared by this scheme has stable properties and known concentration, effectively ensuring the accuracy and repeatability of the detection results.

[0014] Optionally, the fluorescent quantitative PCR reaction system is 25 μL, including: SYBR Mix 12.5 μL, upstream primer 1.0 μL, downstream primer 1.0 μL, DNA template 2.5 μL, sterile deionized water 8.0 μL.

[0015] Optionally, the concentration of the upstream primer and the downstream primer is 10-20 μmol / L.

[0016] By employing the above technical solution, the application optimizes the concentration of the upstream primer and the downstream primer to 10-20 μmol / L, which can obtain higher amplification efficiency on the premise of ensuring amplification specificity, thereby realizing the improvement of detection sensitivity.

[0017] Optionally, the conditions for the fluorescent quantitative PCR are: 95℃ pre-denaturation for 30s; then 95℃ denaturation for 3s, 60℃ annealing and extension for 10s, for 40 cycles.

[0018] Optionally, the concentration of the ampicillin is 80-120 µg / mL.

[0019] In summary, the present application includes at least one of the following beneficial technical effects: 1. By designing primer sets targeting multiple conserved regions in the Langya Henipavirus genome, multi-target detection is achieved, effectively avoiding false negatives due to mutations in a single target, and improving sensitivity. In addition, all primer sets have good amplification efficiency and linear correlation at a unified optimized melting temperature; 2. By cloning the viral genome into a pUC57 vector and amplifying it in E. coli, a high-purity, high-concentration recombinant plasmid is prepared, which is used as a standard for fluorescent quantitative PCR. On this basis, specific primers are used for fluorescent quantitative PCR, and the presence or absence of Langya Henipavirus in the sample can be determined by analyzing the fluorescence amplification signal. The standard prepared in this scheme has stable properties and known concentration, effectively ensuring the accuracy and repeatability of the detection results; 3. By optimizing the concentration of upstream and downstream primers to 10-20 μmol / L, higher amplification efficiency can be achieved while ensuring amplification specificity, thereby improving detection sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the amplification efficiency and linear relationship diagram of the amplification product obtained by using primer set A in embodiment 1 of the present application; Figure 2 is the amplification efficiency and linear relationship diagram of the amplification product obtained by using primer set B in embodiment 2 of the present application; Figure 3 is the standard curve diagram of primer set A at a primer concentration of 20 µM in embodiment 3 of the present application; Figure 4 is the standard curve diagram of primer set B at a primer concentration of 20 µM in embodiment 4 of the present application; Figure 5 is the standard curve diagram of the amplification product obtained by using saliva instead of double distilled water in embodiment 5 of the present application; Figure 6 is the standard curve diagram of the amplification product obtained by using saliva instead of double distilled water in embodiment 6 of the present application. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be described below, and it is obvious that the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0022] The application discloses a primer group A for detecting Langya henipavirus, and at least one of primer group N, primer group M, and primer group P / V / W / C is included. The primer group N includes an upstream primer N-F and a downstream primer N-R, and the sequence list is shown as SEQ ID NO. 1 and SEQ ID NO. 2 respectively; the primer group M includes an upstream primer M-F and a downstream primer M-R, and the sequence list is shown as SEQ ID NO. 5 and SEQ ID NO. 6 respectively; and the primer group P / V / W / C includes an upstream primer P / V / W / C-F and a downstream primer P / V / W / C-R, and the sequence list is shown as SEQ ID NO. 17 and SEQ ID NO. 18 respectively.

[0023] The application discloses a primer group B for detecting Langya henipavirus, and at least one of primer group sg-N, primer group sg-M, primer group sg-L, primer group sg-P / V / W / C-1, and primer group sg-P / V / W / C-2 is included. The primer group sg-N includes an upstream primer sg-N-F and a downstream primer sg-N-R, and the sequence list is shown as SEQ ID NO. 3 and SEQ ID NO. 4 respectively; the primer group sg-M includes an upstream primer sg-M-F and a downstream primer sg-M-R, and the sequence list is shown as SEQ ID NO. 7 and SEQ ID NO. 8 respectively; the primer group sg-L includes an upstream primer sg-L-F and a downstream primer sg-L-R, and the sequence list is shown as SEQ ID NO. 25 and SEQ ID NO. 26 respectively; the primer group sg-P / V / W / C-1 includes an upstream primer sg-P / V / W / C-1-F and a downstream primer sg-P / V / W / C-1-R, and the sequence list is shown as SEQ ID NO. 19 and SEQ ID NO. 20 respectively; and the primer group sg-P / V / W / C-2 includes an upstream primer sg-P / V / W / C-2-F and a downstream primer sg-P / V / W / C-2-R, and the sequence list is shown as SEQ ID NO. 21 and SEQ ID NO. 22 respectively.

[0024] The application designs a kit for detecting Langya henipavirus, and the kit includes the primer group A for detecting Langya henipavirus or the primer group B for detecting Langya henipavirus.

[0025] The application designs a method for detecting Langya henipavirus, and the method is not for the purpose of diagnosing a disease, and includes the following steps. S1, obtaining a Langya henipavirus genome, cloning the Langya henipavirus genome to a pUC57 plasmid vector to obtain a recombinant plasmid; S2, transform the recombinant plasmid to E. coli Top10 cells, culture in the medium containing 80-120 μg / mL ampicillin, then extract plasmid DNA, purify, and obtain the purified recombinant plasmid.

[0026] S3, perform fluorescent quantitative PCR on the purified recombinant plasmid using primer group A for detecting Langya Henipavirus or primer group B for detecting Langya Henipavirus, the reaction system is 25 μL, including: SYBR Mix 12.5 μL, upstream primer 1.0 μL, downstream primer 1.0 μL, DNA template 2.5 μL, sterile deionized water 8.0 μL; the condition is: 95℃ pre-denaturation for 30 s; then 95℃ denaturation for 3 s, 60℃ annealing and extension for 10 s, for 40 cycles. According to the fluorescence amplification signal of real-time fluorescent quantitative PCR, it is determined whether the sample contains Langya Henipavirus.

[0027] The reagents used in the embodiments of the present application can be obtained by marketing, wherein: pUC57 plasmid vector, Shanghai Sang Biotechnology Co., Ltd.; SYBR premix, Hieff UNICON® SYBR Green, Yixing Biotechnology (Shanghai) Co., Ltd.; LB medium, Shanghai Weidi Biotechnology Co., Ltd.; Ampicillin, Shanghai Yingxin Laboratory Equipment Co., Ltd.; Silica gel column kit, Tissue DNA Kit D3396, Guangzhou Feiyang Biological Engineering Co., Ltd.; Ultra-micro UV-visible spectrophotometer, model NanoDrop One, Thermo Fisher Scientific Corporation. Specific embodiments

[0028] Preparation Example 1 Preparation of saliva sample: collect fresh saliva of the patient by using sterile sampling cotton, place it in a saliva collection tube, add inactivated nuclease, and mix uniformly to obtain the saliva sample. Example 1

[0029] The genomic sequence of Langya henipavirus (Accession No. OM101125.1) was obtained from NCBI Genebank, and the relatively conserved M region, N region, G region, F region, L region, P / V / W / C region were selected as the amplified gene fragments. The primers were designed according to the target gene fragments, and the Primer-BLAST software was used for design. The primers with optimal GC content (about 50%) and melting temperature (about 56°C) were designed, and all primers were synthesized by Shanghai Sangon Biological Technology Co., Ltd. The designed primers were primer group N, primer group M, primer group G, primer group F, primer group L, and primer group P / V / W / C. All primers are shown in Tables 1-2. The Langya henipavirus genome (M region, N region, G region, F region, L region, P / V / W / C-V protein region, P / V / W / C-W protein region, P / V / W / C-C protein region) was cloned into the pUC57 plasmid vector to obtain a recombinant plasmid, and the length of the recombinant plasmid is shown in Table 1. Then the recombinant plasmid was transformed into E. coli Top10 cells, cultured in LB medium containing 100 µg / mL ampicillin, and then the plasmid DNA was extracted using a silica gel column kit, purified, and the purified recombinant plasmid was obtained. The recombinant plasmid was diluted by 10-fold dilution method, and the copy number was diluted from 10 8 to 10 3 , and three repeated detections were performed. The purified recombinant plasmid was subjected to fluorescent quantitative PCR using the above primer groups. The fluorescent quantitative PCR reaction system was 25 μL, including: SYBR premix 12.5 μL, upstream primer 1.0 μL (10 μmol / L), downstream primer 1.0 μL (10 μmol / L), DNA template 2.5 μL, sterile deionized water 8.0 μL; the conditions were: 95°C pre-denaturation for 30 s; then 95°C denaturation for 3 s, 60°C annealing and extension for 10 s, for 40 cycles, to obtain the amplification product.

[0030] The plasmid concentration was determined using an ultramicro UV-visible spectrophotometer, and the DNA copy number was calculated. The calculation formula was: copy number = (DNA concentration / (plasmid length × 660)) × 6.022 × 10 23 . Wherein, the DNA concentration unit is ng / uL, the plasmid length unit is bp, and 660 is the average base molecular weight (g / mol). The specific plasmid length is shown in Table 1.

[0031] The standard curve was drawn according to the ct value and copy number of fluorescent quantitative PCR, and the amplification efficiency was calculated. The amplification efficiency = -1 + 10 （-1 / 斜率） . Wherein, the slope is the slope of the standard curve. Example 2

[0032] The genomic sequence of Langya henipavirus was obtained from NCBI Genebank, and the relatively conserved M region, N region, G region, F region, L region, P / V / W / C region were selected as the amplified gene fragments. The primers were designed according to the target gene fragments, and the Primer-BLAST software was used for primer design. The primers with optimal GC content (about 50%) and melting temperature (about 60°C) were designed. All primers were synthesized by Shanghai Shengong Biotechnology Co., Ltd. The designed primers were primer group sg-N, primer group sg-M, primer group sg-G, primer group sg-F, primer group sg-L, primer group sg-P / V / W / C-1, primer group sg-P / V / W / C-2, and all primers are shown in Tables 1-2. The Langya henipavirus genome (M region, N region, G region, F region, L region, P / V / W / C-V protein region, P / V / W / C-W protein region, P / V / W / C-C protein region) was cloned into the pUC57 plasmid vector to obtain a recombinant plasmid, and the length of the recombinant plasmid is shown in Table 1. Then the recombinant plasmid was transformed into E. coli Top10 cells, cultured in medium containing 100 µg / mL ampicillin, and then the plasmid DNA was extracted, purified, and the purified recombinant plasmid was obtained. The purified recombinant plasmid was subjected to fluorescent quantitative PCR using the above primer groups, and the reaction system was 25 μL, including: SYBR premix 12.5 μL, upstream primer 1.0 μL (10 μmol / L), downstream primer 1.0 μL (10 μmol / L), DNA template 2.5 μL, sterile deionized water 8.0 μL; The conditions are: 95°C pre-denaturation for 30s; then 95°C denaturation for 3s, 60°C annealing and extension for 10s, for 40 cycles, to obtain the amplification product.

[0033] The amplification efficiency and R 2 of the amplification product obtained in Example 1-2 were calculated, and the detection results are shown in Figures 1-2

[0034] Table 1 Length of plasmid for copy number determination

[0035] Table 2 Primer sequences (SEQ ID NO: 1-26) for detecting Langya henipavirus

[0036] As can be seen from Example 1-2, Figures 1-2 and Tables 1-2, the primer groups designed in the present application for fluorescent quantitative PCR have an amplification efficiency in the range of 91%-106%, and R 2 ​Above 0.9833, it indicates that the primer sets (primer set N, primer set M, primer set P / V / W / C, primer set sg-N, primer set sg-M, primer set sg-L, primer set sg-P / V / W / C-1 and primer set sg-P / V / W / C-2) designed in the application all achieve efficient amplification in the dilution range, and exhibit strong linear relationship and stable performance. In addition, the amplification products obtained by performing the fluorescent quantitative PCR by using primer set G, primer set F, primer set L, primer set sg-G and primer set sg-F fail to obtain the standard curve due to non-specific amplification. Example 3

[0037] The difference between the present example and Example 1 is that, in the present example, 10 μmol / L of the upstream primer 1.0 μL in Example 1 is replaced by 20 μmol / L of the upstream primer 1.0 μL, and 10 μmol / L of the downstream primer 1.0 μL in Example 1 is replaced by 20 μmol / L of the downstream primer 1.0 μL. Example 4

[0038] The difference between the present example and Example 2 is that, in the present example, 10 μmol / L of the upstream primer 1.0 μL in Example 2 is replaced by 20 μmol / L of the upstream primer 1.0 μL, and 10 μmol / L of the downstream primer 1.0 μL in Example 2 is replaced by 20 μmol / L of the downstream primer 1.0 μL.

[0039] The amplification efficiency and R 2 of the amplification products obtained in Examples 3-4 are calculated. Figures 3-4

[0040] It can be known from Examples 3-4 and Figures 3-4 that, for the sg-L gene target in primer set B, when the concentrations of the upstream and downstream primers are 10 μmol / L, the initial amplification is slightly delayed; but after the primer concentration is increased to 20 μmol / L, the amplification curve is improved, and the melting curve is still single-peak, indicating that the optimization does not introduce non-specific amplification while improving the sensitivity. Example 5

[0041] The difference between the present example and Example 1 is that, in the present example, the sterile deionized water of the same mass in Example 1 is replaced by the saliva sample obtained in Preparation Example 1. Example 6

[0042] The difference between the present example and Example 2 is that, in the present example, the sterile deionized water of the same mass in Example 2 is replaced by the saliva sample obtained in Preparation Example 1.

[0043] The amplification efficiency and R 2 ​The results are shown in Table 1. Figures 5-6

[0044] As can be seen from Examples 5-6 and Figures 5-6 It can be seen that, when saliva samples are used instead of sterile deionized water for fluorescence quantitative PCR, all gene targets can be detected, and no inhibition phenomenon is observed. The ct value only changes by 0.5-1.2 cycles, but still maintains a linear relationship and amplification efficiency.

[0045] The above are preferred embodiments of the present application, and are not intended to limit the protection scope of the present application, so: all equivalent changes made according to the structure, shape, principle of the present application should be covered within the protection scope of the present application.​

Claims

1. A primer set A for detecting Lanyai heungni papaya virus, characterized in that, at least one of primer group N, primer group M, primer group P / V / W / C; the primer group N comprises upstream primer N-F and downstream primer N-R, and their sequence lists are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2; the primer group M comprises upstream primer M-F and downstream primer M-R, and their sequence lists are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6; the primer group P / V / W / C comprises upstream primer P / V / W / C-F and downstream primer P / V / W / C-R, and their sequence lists are respectively shown as SEQ ID NO. 17 and SEQ ID NO.

18.

2. A primer set B for detecting Lanyai heinipavirus, characterized in that, at least one of primer group sg-N, primer group sg-M, primer group sg-L, primer group sg-P / V / W / C-1, primer group sg-P / V / W / C-2; the primer group sg-N comprises upstream primer sg-N-F and downstream primer sg-N-R, and their sequence lists are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4; the primer group sg-M comprises upstream primer sg-M-F and downstream primer sg-M-R, and their sequence lists are respectively shown as SEQ ID NO. 7 and SEQ ID NO. 8; the primer group sg-L comprises upstream primer sg-L-F and downstream primer sg-L-R, and their sequence lists are respectively shown as SEQ ID NO. 25 and SEQ ID NO. 26; the primer group sg-P / V / W / C-1 comprises upstream primer sg-P / V / W / C-1-F and downstream primer sg-P / V / W / C-1-R, and their sequence lists are respectively shown as SEQ ID NO. 19 and SEQ ID NO. 20; the primer group sg-P / V / W / C-2 comprises upstream primer sg-P / V / W / C-2-F and downstream primer sg-P / V / W / C-2-R, and their sequence lists are respectively shown as SEQ ID NO. 21 and SEQ ID NO.

22.

3. A kit for detecting Lyoni hengi henipavirus, characterized by, the primer group A for detecting the Langya Henipavirus according to claim 1 or the primer group B for detecting the Langya Henipavirus according to claim 2.

4. A method for validating a primer set for Lanyanghe hepevirus, characterized by, comprising the following steps: S1, obtaining a Langya Henipavirus genome, cloning the Langya Henipavirus genome into a pUC57 plasmid vector to obtain a recombinant plasmid; S2, transforming the recombinant plasmid into E. coli Top10 cells, culturing in a culture medium containing ampicillin, then extracting plasmid DNA, purifying to obtain a purified recombinant plasmid; S3, performing fluorescent quantitative PCR on the purified recombinant plasmid using the primer group A for detecting the Langya Henipavirus according to claim 1 or the primer group B for detecting the Langya Henipavirus according to claim 2, and determining whether the sample contains the Langya Henipavirus according to the fluorescent amplification signal of the real-time fluorescent quantitative PCR.

5. The method for verifying the primer set for Lanyai henipavirus according to claim 4, characterized in that, the fluorescent quantitative PCR reaction system is 25 μL, The method comprises: 12.5 μL of SYBR premix, 1.0 μL of upstream primer, 1.0 μL of downstream primer, 2.5 μL of DNA template, and 8.0 μL of sterile deionized water.

6. The method for verifying the primer set for Lanyai henipavirus according to claim 5, characterized in that, The concentration of the upstream primer and the downstream primer is 10-20 μmol / L.

7. The method for verifying the primer set for Lanyai henipavirus according to claim 4, characterized in that, The condition of the fluorescent quantitative PCR is: 95 ℃ pre-denaturation for 30 s; then 95 ℃ denaturation for 3 s, 60 ℃ annealing and extension for 10 s, for 40 cycles.

8. The method for verifying the primer set for Lanyai henipavirus according to claim 4, characterized in that, The concentration of the ampicillin is 80-120 µg / mL.

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