Sequence, kit and detection method for nipah virus nucleic acid detection

Abstract

The invention aims to provide the nipah virus nucleic acid detection sequence, the kit and the detection method, the detection time is short, only 35 minutes are needed, the specificity is good, the sensitivity is high, the anti-interference capability is strong, and the nipah virus nucleic acid detection sequence, the kit and the detection method are suitable for early detection and case discovery of nipah virus infection and can better prevent and control nipah viruses. The primer comprises a primer and crRNA, wherein the primer comprises a forward primer and a reverse primer. The invention is applied to the technical field of virus nucleic acid detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of virus nucleic acid detection, and particularly relates to a sequence, a kit and a detection method for detecting nucleic acid of a Nipah virus. BACKGROUND

[0002] The Nipah virus is a single-stranded negative-sense RNA virus of the Henipavirus genus of the Paramyxoviridae family, is an acute and highly lethal zoonotic virus, bats are its natural host, and pigs and humans can also be infected, and the infected person initially shows flu-like symptoms such as fever, headache, and vomiting, which can rapidly develop into severe meningitis, respiratory failure, etc., and the mortality rate is as high as 40% to 75%. There is currently no vaccine and effective treatment for Nipah virus disease.

[0003] Currently, the detection methods for the Nipah virus mainly include serological detection and polymerase chain reaction (PCR) detection technology. The serological detection method is difficult to detect cases in the early stage of infection, and specific antibodies can be detected only after 5-7 days of onset. Nucleic acid detection has the characteristics of high sensitivity and can be detected in the early stage of disease, and the current main means is PCR detection. PCR technology is a molecular biology technology that realizes DNA amplification in vitro through temperature control, and its core includes three stages of denaturation, annealing, and extension, and is widely used in the fields of medical diagnosis, gene research, and quality control. However, PCR requires professional electrophoresis equipment, fluorescence quantitative PCR equipment, and has high requirements for the operating environment and personnel level, and the result output time is relatively long, and has certain limitations.

[0004] A kit for detecting Nipah virus nucleoprotein antibodies and its application are disclosed in Chinese Patent No. CN118566502A, which is suitable for large-scale serological screening. However, the serological detection method is difficult to detect cases in the early stage of infection, and specific antibodies can be detected only after 5-7 days of onset. Therefore, it is necessary to provide a sequence, a kit and a detection method for detecting nucleic acid of a Nipah virus, which has a short detection time of only 35 minutes, good specificity, high sensitivity, strong anti-interference ability, is suitable for early detection of Nipah virus infection and case finding, and can better prevent and control the Nipah virus. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the shortcomings of the prior art, and a sequence, a kit and a detection method for detecting nucleic acid of a Nipah virus are provided, which has a short detection time of only 35 minutes, good specificity, high sensitivity, strong anti-interference ability, is suitable for early detection of Nipah virus infection and case finding, and can better prevent and control the Nipah virus.

[0006] The technical scheme adopted by the present application is: the present application comprises primers and crRNA, the primers comprise forward primers and reverse primers, and the base sequences of the forward primers, the reverse primers and the crRNA are respectively as follows: The forward primer is as follows: Niv-F: 5'-TAATACGACTCACTATAGGGTGATGCTACTCTACAGAGAAATTGGCCCAAG-3', SEQ ID NO: 1; which is located at positions 900-930 of GenBank reference sequence JN808862.1) 103-1 = 102 104-2; The reverse primer is as follows: Niv-R: 5'-ACATAGGTTCAAGATAACCACGATTGATGTT-3', SEQ ID NO: 2; which is located at positions 1055-1085 of GenBank reference sequence JN808862.1; The crRNA is as follows: 5'-gauuuagacuaccccaaaaacgaaggggacuaaaacCUGAAUUGAUUCUUCAAGAAGCACC AUA-3', SEQ ID NO: 3; which is located at positions 940-967 of GenBank reference sequence JN808862.1.

[0007] From the above scheme, the application establishes a Nipah virus nucleic acid detection method based on RPA-Crispr / Cas13a technology. CRISPR (clustered regularly interspaced short palindromic repeats) is a group of DNA sequences found in prokaryotes such as bacteria and archaea. The CRISPR-Cas system mainly consists of a leader sequence, a CRISPR locus, and CRISPR-related genes encoding Cas (CRISPR-associated proteins). Under the action of crRNA targeted recognition of foreign genes, the endonuclease activity of Cas protein is activated to cut foreign genetic material and achieve immune effect. Since 2011 when the system was discovered, researchers have continuously discovered new protein members. Currently, the CRISPR / Cas system mainly has two types with more than thirty subtypes. Among them, Cas9, Cas12, Cas13, etc. in the second type have shown great application prospects in the field of molecular detection. In 2019, Zhang Feng's team combined isothermal amplification technology with CRISPR / Cas system to develop SHERLOCK platform, realizing rapid detection of pathogens. The application uses the above primers and crRNA to realize a kit for Nipah virus detection with high repeatability, high specificity, and high sensitivity. The detection time is short; it can be applied to clinical and port routine detection and disease prevention and control fields.

[0008] One preferred scheme is that the kit comprises RPA reaction reagents and Crispr reaction reagents. The RPA reaction reagents comprise buffer A, 20uM primer Niv-F, 20uM primer Niv-R, Starter, reaction tube Tube, wherein the primer Niv-F is a nucleotide sequence as shown in SEQ ID NO: 1, and the primer Niv-R is a nucleotide sequence as shown in SEQ ID NO: 2. The Crispr reaction reagents comprise Cleavage Buffer, Trans Mix, T7 RNA Polymerase, Cas13a Protein (2uM), crRNA (0.4uM), Reporter (4uM), Nuclease-free Water, wherein the crRNA is a nucleotide sequence as shown in SEQ ID NO: 3.

[0009] One preferred scheme is that the kit comprises RPA reaction reagents and Crispr reaction reagents, characterized in that the positive control is an in vitro transcribed RNA fragment of Nipah virus.

[0010] One preferred embodiment is that the negative control is sterile normal saline, which is extracted simultaneously with the specimen as a negative control during nucleic acid extraction.

[0011] One preferred embodiment is that the blank control is nuclease-free ultrapure water.

[0012] One preferred embodiment is that the preparation method of the positive control comprises the following steps: Step A1, synthesizing a plasmid according to the reference sequence JN808862.1 downloaded from GenBank; Step A2, obtaining single-stranded RNA by in vitro transcription using T7 RNA polymerase, and removing DNA molecules after transcription by DNase I digestion; Step A3, performing column purification using QIAGEN RNeasy MiniElute Cleanup kit; Step A4, determining the concentration of the purified RNA using a micro-UV spectrophotometer, and calculating the copy number by its molecular weight; Step A5, after aliquoting, store at -80°C, as a positive control for the kit.

[0013] One preferred embodiment is that the detection method comprises the following steps: Step B1, extracting the RNA of the sample, i.e., the template RNA: the kit does not provide RNA sample extraction reagents, and the user can select appropriate commercial kits to extract viral nucleic acids according to the sample type; Step B2, preparing the RPA reaction solution according to the following method: the reaction volume is 50 uL; add the reaction mixture to each reaction tube Tube, including 29.4 uL of buffer A, 0.4 uM of each primer (Niv-F, Niv-R,), 2.5 uL of Starter, and 10 uL of RNA template. After mixing, centrifuge for a few seconds, and place in a water bath or metal bath or PCR instrument at 39°C for 10 min; Step B3, preparing the Crispr reaction solution for machine detection: the reaction volume is 20 uL; the mixture includes Cleavage Buffer 2 uL, TransMix 4 uL, T7 RNA Polymerase 0.5 uL, Cas13a Protein (2 uM) 0.6 uL, crRNA (0.4 uM) 1.5 uL, Reporter (4 uM) 0.6 uL, the product of the first reaction 5 uL, Nuclease-free Water 5.8 uL, mix well, and place in a fluorescent PCR instrument at 37°C for 25 min, collecting the fluorescence of the FAM channel every minute; Step B4, Result Judgment: First, the quality control system should be judged, that is, the amplification curve of the positive control in each fluorescence channel should show a standard S-shaped curve, while the negative control and blank control should not show a typical S-shaped curve. When the quality control system is effective, the results of the test samples are judged as follows: if the FAM channel of the test sample has an obvious S-shaped curve, it can be judged as positive for Nipah virus nucleic acid; if the FAM channel of the test sample does not show fluorescence amplification, it can be judged as negative for Nipah virus nucleic acid.

[0014] In a preferred embodiment, the detection method includes sensitivity analysis. The method for sensitivity analysis is as follows: (1) Sample processing: The viral RNA transcribed in vitro was used as the template for detection. The concentration of purified RNA was measured by a micro-ultraviolet spectrophotometer. The copy number of the initial RNA template was calculated by molecular weight. Then, it was serially diluted 10-fold to single copy number, for a total of 4 gradients. The copy number was 10^3 copies / uL to 1 copies / uL. (2) Detection: The reaction solution is prepared and detected using the detection method of claim 7, and the minimum template copy number that the kit can detect is analyzed; (3) Results: The test results show that the detection limit of the kit is 1 copy / uL and the total detection time is 35 min. It can be seen that the test kit is fast and highly sensitive, and can play an important role in rapid emergency detection.

[0015] In a preferred embodiment, the detection method includes repeatability analysis. The method for repeatability analysis is as follows: (1) Sample processing: The viral RNA transcribed in vitro was used as the template for detection at a concentration of 1 copy / uL; (2) Detection: The reaction solution was prepared using the detection method described in claim 7, and the detection was repeated 10 times. Then, the reaction tube was placed on a fluorescence PCR instrument for detection. After the reaction was completed, the fluorescence curve was observed, and the repeatability of the detection by the kit described in this invention was analyzed. (3) Results: The test results showed that the kit was positive 10 times at a concentration of 1 copies / uL, which met the expected results; it can be seen that the test kit has good repeatability.

[0016] In a preferred embodiment, the detection method includes specificity analysis. The method for the specificity analysis is as follows: (1) Sample processing: a group of clinical positive samples of respiratory disease related pathogens are selected, including influenza A H1N1 virus, influenza A H3N2 virus, influenza B virus, new coronavirus, respiratory adenovirus, respiratory syncytial virus, rhinovirus, Haemophilus influenzae, Legionella, Streptococcus pneumoniae, Mycoplasma pneumoniae, monkeypox virus, and in vitro transcribed Nipah virus RNA (10^5 copies / uL); (2) Detection: the above 13 samples are detected using the detection kit and the method of claim 7, to observe whether the kit will have non-specific detection results; (3) Results: according to the analysis of the fluorescence spectrum of the amplification of the detection kit, the detection results of the kit are positive only for the sample containing Nipah virus RNA, and the detection results of the other 12 pathogens and the negative control are negative, proving that the method has good specificity. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is the detection result graph of the application applied to sensitivity analysis, from top to bottom, 10^3 copies / uL~1 copies / uL Nipah virus RNA sample; Figure 2 is the detection result graph of the application applied to repeatability analysis, 10 curves are the detection result graphs of 1 copies / uL Nipah virus RNA sample repeated 10 times; Figure 3 is the detection result graph of the application applied to specificity analysis. DETAILED DESCRIPTION

[0018] Example 1 Specificity primer design: By collecting 93 Nipah virus genome sequences included in GenBank, homologous comparison is performed, and key elements such as variable region, conserved region, and specific region are analyzed. The length of the RPA primer is generally 30 bp~35 bp, and special sequences such as long string of poly-pu and poly-py should not appear in the primer. The GC content is preferably between 40%~60%, the design of crRNA should not overlap with the RPA primer, and according to the above principles, a plurality of RPA primers and crRNAs are designed. After the designed primers are synthesized back and then screened and verified by samples, the following RPA primers and crRNAs are finally selected by the application.

[0019] The application includes primers and crRNA, the primers include forward primers and reverse primers, and the base sequences of the forward primers, the reverse primers, and the crRNA are as follows: The forward primer: Niv-F:5'-TAATACGACTCACTATAGGGTGATGCTACTCTACAGAGAAATTGGCCCAAG-3', SEQ ID NO: 1; which is at positions 900-930 of GenBank Reference Sequence JN808862.1) 103-1 = 102 104-2; the reverse primer: Niv-R:5'-ACATAGGTTCAAGATAACCACGATTGATGTT-3', SEQ ID NO: 2; which is at positions 1055-1085 of GenBank Reference Sequence JN808862.1; the crRNA: 5'-gauuuagacuaccccaaaaacgaaggggacuaaaacCUGAAUUGAUUCUUCAAGAAGCACC AUA-3', SEQ ID NO: 3; which is at positions 940-967 of GenBank Reference Sequence JN808862.1.

[0020] In this embodiment, by collecting the genomic sequence of the Nipah virus included in GenBank, homologous alignment is performed, and key elements such as variable region, conserved region, specific region, etc. are analyzed. The length of the general RPA primer is 30 bp-35 bp. Special sequences such as long string of poly-pu, poly-py should not appear in the primer. The GC content is preferably between 40%-60%. The design of crRNA cannot overlap with the RPA primer. According to the above principles, a plurality of RPA primers and crRNA are designed, and suitable RPA primers and crRNA are screened out through testing. Adjust the concentrations of primers, crRNA, enzymes, etc. After optimization, the RPA-Crispr / Cas13a reaction system is established. The first reaction mixture is added to each reaction tube Tube 29.4 uL buffer A, each primer (Niv-F, Niv-R,) 0.4 uM, 2.5 uL Starter and 10 uL RNA template. Mix well and put into water bath or metal bath or PCR instrument 39℃ 10 min. The second reaction mixture contains Cleavage Buffer 2uL, TransMix 4uL, T7 RNA Polymerase 0.5uL, Cas13a Protein (2uM) 0.6uL, crRNA (0.4uM) 1.5uL, Reporter (4uM) 0.6uL, the product of the first reaction 5uL, Nuclease-free Water 5.8uL, mix well and put into PCR instrument 37℃ 25 min, collect FAM channel fluorescence every minute.

[0021] Example Two Kit composition and detection method for detecting Nipah virus based on RPA-Crispr / Cas13a technology: The kit comprises RPA reaction reagents and Crispr reaction reagents; The RPA reaction reagents comprise buffer A, 20uM primer Niv-F, 20uM primer Niv-R, Starter, reaction tube Tube, wherein the primer Niv-F is a nucleotide sequence as shown in SEQ ID NO: 1, and the primer Niv-R is a nucleotide sequence as shown in SEQ ID NO: 2; The Crispr reaction reagents comprise Cleavage Buffer, Trans Mix, T7 RNA Polymerase, Cas13a Protein (2uM), crRNA (0.4uM), Reporter (4uM), Nuclease-free Water, wherein the crRNA is a nucleotide sequence as shown in SEQ ID NO: 3.

[0022] In the present embodiment, the kit comprises RPA reaction reagents and Crispr reaction reagents, characterized in that the positive control is an in vitro transcribed RNA fragment of Nipah virus.

[0023] In the present embodiment, the negative control is sterile normal saline, which is extracted simultaneously with the specimen during nucleic acid extraction and used as a negative control.

[0024] In the present embodiment, the blank control is nuclease-free ultrapure water.

[0025] In the present embodiment, the preparation method of the positive control comprises the following steps: Step A1, synthesizing a plasmid according to the reference sequence JN808862.1 downloaded from GenBank; Step A2, obtaining single-stranded RNA by in vitro transcription using T7 RNA polymerase, and removing DNA molecules in the single-stranded RNA by DNase I digestion after transcription; Step A3, performing column purification using QIAGEN RNeasy MiniElute Cleanup kit; Step A4, determining the concentration of the purified RNA by using a micro ultraviolet spectrophotometer, and calculating the copy number by the molecular weight; Step A5, after aliquoting, storing at -80℃, as the positive control of the kit.

[0026] In the present embodiment, the detection method comprises the following steps: Step B1, extracting the RNA of the sample, i.e. the template RNA: the kit of the present application does not provide RNA sample extraction reagents, and the user can select appropriate commercial kits to extract viral nucleic acids according to the sample type; Step B2, preparing the RPA reaction solution according to the following method: the reaction volume is 50 μL; adding the reaction mixture to each reaction tube Tube, including 29.4 uL buffer A, 0.4 uM of each primer (Niv-F, Niv-R,), 2.5 uL Starter and 10 uL RNA template. After mixing, centrifuge for a few seconds, and place in a water bath or metal bath or PCR instrument at 39℃ for 10 min; Step B3, prepare the Crispr reaction solution for machine detection: the reaction volume is 20 μL; the mixture contains Cleavage Buffer 2 uL, Trans Mix 4 uL, T7 RNA Polymerase 0.5 uL, Cas13a Protein (2 uM) 0.6 uL, crRNA (0.4 uM) 1.5 uL, Reporter (4 uM) 0.6 uL, the product of the first reaction 5 uL, Nuclease-free Water 5.8 uL, mix well and put into the fluorescence PCR instrument at 37°C for 25 min, collect the fluorescence of the FAM channel every minute; Step B4, result determination: first, judge the quality control system, that is, the amplification curve of the positive control of each fluorescence channel presents a standard S-shaped curve, and the negative control and blank control have no typical S-shaped curve; When the quality control system is effective, the results of the sample to be tested are determined. When the sample to be tested has a significant S-shaped curve in the FAM channel, it is determined that the Nipah virus nucleic acid is positive. When the sample to be tested has no fluorescence amplification phenomenon in the FAM channel, it is determined that the Nipah virus nucleic acid is negative.

[0027] Notes: No powder gloves should be used throughout the experiment. In order to avoid cross contamination in the experiment, first add the blank and negative control, then add the sample to be tested, and finally add the positive control.

[0028] Example Three Sensitivity analysis of the kit for detecting Nipah virus based on RPA-Crispr / Cas13a technology, as shown in Figure 1 The method of the sensitivity analysis is as follows: (1) Sample processing: the above in vitro transcribed viral RNA is used as the template for detection, the concentration of the purified RNA is determined by using a micro ultraviolet spectrophotometer, the initial RNA template copy number is calculated by molecular weight, and then it is diluted by 10 times gradient to the individual copy number, a total of 4 gradients, the copy number is: 10^3 copies / uL~1 copies / uL; (2) Detection: the detection method of claim 7 is used for reaction solution preparation and detection, and the lowest limit of template copy number that can be detected by the kit is analyzed; (3) Results: the detection results show that the detection lower limit of the kit is 1 copies / uL, and the total detection time is 35 min. It can be seen that the detection kit is rapid and has high sensitivity, and can play an important role in rapid emergency detection.

[0029] Example Four Reproducibility analysis of the kit for detecting Nipah virus based on RPA-Crispr / Cas13a technology, as shown inFigure 2 As shown in the figure, the method of the repeatability analysis is as follows: (1) Sample processing: the above in vitro transcribed viral RNA is used as a template for detection, with a concentration of 1 copies / uL; (2) Detection: the reaction solution is prepared by using the detection method of claim 7, and the detection is repeated 10 times, and then the reaction tube is placed on the fluorescence PCR instrument for detection; after the reaction is completed, the fluorescence curve pattern is observed, and the repeatability of the kit detection is analyzed; (3) Results: the detection results show that the kit is positive for 10 times at a concentration of 1 copies / uL, which meets the expected results; it can be seen that the detection kit has good repeatability.

[0030] Example five The specificity analysis of the kit for detecting Nipah virus based on RPA-Crispr / Cas13a technology is as follows: Figure 3 As shown in the figure, the method of the repeatability analysis is as follows: (1) Sample processing: a group of clinical positive samples of respiratory disease related pathogens are selected, including influenza A H1N1 virus, influenza A H3N2 virus, influenza B virus, new coronavirus, respiratory adenovirus, respiratory syncytial virus, rhinovirus, Haemophilus influenzae, Legionella, Streptococcus pneumoniae, Mycoplasma pneumoniae, monkeypox virus, and in vitro transcribed Nipah virus RNA (105 copies / uL); (2) Detection: the above 13 samples are detected by using the detection kit and the method of claim 7, and whether the kit will have non-specific detection results is observed; (3) Results: according to the fluorescence spectrum of the amplification of the detection kit, the detection results of the kit for the sample containing Nipah virus RNA are positive, and the detection results of the other 12 kinds of pathogens and negative control are negative, which proves that the method has good specificity.

[0031] Although the embodiments of the present application are described in actual schemes, but do not constitute a limitation on the meaning of the present application, for those skilled in the art, according to the modification of the embodiments thereof and the combination with other schemes are obvious.

Claims

1. A nucleic acid detection sequence for Nipah virus, comprising primers and crRNA, characterized in that: The primers include a forward primer and a reverse primer, and the base sequences of the forward primer, the reverse primer, and the crRNA are represented as follows: The forward primer: Niv-F:5'-TAATACGACTCACTATAGGGTGATGCTACTCTACAGAGAAATTGGCCCAAG-3', SEQ IDNO: 1; Its position in the GenBank reference sequence JN808862.1 is (900~930) 103-1=102 104-2; The reverse primer: Niv-R: 5'-ACATAGGTTCAAGATAACCACGATTGATGTT-3', SEQ ID NO: 2; Its position in the GenBank reference sequence JN808862.1 is 1055~1085; The crRNA: 5'-gauuuagacuaccccaaaaacgaaggggacuaaaacCUGAAUUGAUUCUUCAAGAAGCACC AUA-3', SEQ ID NO: 3; Its position in the GenBank reference sequence JN808862.1 is 940~967.

2. A kit comprising the Nipah virus nucleic acid detection sequence as described in claim 1, characterized in that: The kit includes RPA reaction reagents and CRISPR reaction reagents; The RPA reaction reagent includes buffer A, 20 μM primer Niv-F, 20 μM primer Niv-R, starter, and reaction tube, wherein primer Niv-F is the nucleotide sequence shown in SEQ ID NO: 1, and primer Niv-R is the nucleotide sequence shown in SEQ ID NO: 2; The CRISPR reaction reagents include Cleavage Buffer, Trans Mix, T7 RNA Polymerase, Cas13aProtein (2uM), crRNA (0.4uM), Reporter (4uM), and Nuclease-free Water, wherein the crRNA is the nucleotide sequence shown in SEQ ID NO:

3.

3. The reagent kit according to claim 2, characterized in that: The kit includes RPA reaction reagent and CRISPR reaction reagent, characterized in that: the positive control is an RNA fragment transcribed from Nipah virus in vitro.

4. The reagent kit according to claim 2, characterized in that: The negative control was sterile physiological saline, which was extracted simultaneously with the sample during nucleic acid extraction and used as a negative control.

5. The reagent kit according to claim 2, characterized in that: The blank control was ultrapure water without nuclease.

6. The reagent kit according to claim 3, characterized in that: The method for preparing the positive control includes the following steps: Step A1: Synthesize plasmid based on the reference sequence JN808862.1 downloaded from GenBank; Step A2: Use T7 RNA polymerase to perform in vitro transcription to obtain single-stranded RNA, and then digest with DNase I to remove the DNA molecules. Step A3: Perform column purification using the QIAGEN RNeasy MiniElute Cleanup kit; Step A4: Determine the concentration of purified RNA using a micro-ultraviolet spectrophotometer, and calculate the copy number based on its molecular weight; Step A5: After aliquoting, store at -80℃ as a positive control for the kit.

7. A detection method comprising the kit of claim 3, characterized in that: The detection method includes the following steps: Step B1: Extract RNA from the sample, i.e. template RNA: The kit of this invention does not provide RNA sample extraction reagents. Users can select appropriate commercial kits to extract viral nucleic acid according to the sample type. Step B2: Prepare the RPA reaction solution as follows: The reaction volume is 50 μL; add the reaction mixture to each reaction tube containing 29.4 μL of buffer A, 0.4 μM of each primer (Niv-F, Niv-R), 2.5 μL of starter, and 10 μL of RNA template; after mixing, centrifuge for a few seconds, and place in a water bath, metal bath, or PCR instrument at 39℃ for 10 min; Step B3: Prepare the CRISPR reaction solution for instrument detection: The reaction volume is 20 μL; the mixture contains 2 μL of Cleavage Buffer, 4 μL of Trans Mix, 0.5 μL of T7 RNA Polymerase, 0.6 μL of Cas13a Protein (2 μM), 1.5 μL of crRNA (0.4 μM), 0.6 μL of Reporter (4 μM), 5 μL of the product from the first reaction, and 5.8 μL of Nuclease-free Water. After mixing, place the mixture on a fluorescence PCR instrument at 37°C for 25 min, and collect the fluorescence of the FAM channel once per minute. Step B4, Result Judgment: First, the quality control system should be judged, that is, the amplification curve of the positive control in each fluorescence channel should show a standard S-shaped curve, while the negative control and blank control should not show a typical S-shaped curve. When the quality control system is effective, the results of the test samples are judged as follows: if the FAM channel of the test sample has an obvious S-shaped curve, it can be judged as positive for Nipah virus nucleic acid; if the FAM channel of the test sample does not show fluorescence amplification, it can be judged as negative for Nipah virus nucleic acid.

8. The detection method according to claim 7, characterized in that: The detection method includes sensitivity analysis, and the sensitivity analysis method is as follows: (1) Sample processing: The viral RNA transcribed in vitro was used as the template for detection. The concentration of purified RNA was measured by a micro-ultraviolet spectrophotometer. The copy number of the initial RNA template was calculated by molecular weight. Then, it was serially diluted 10-fold to single copy number, for a total of 4 gradients. The copy number was 10^3 copies / uL to 1 copies / uL. (2) Detection: The reaction solution is prepared and detected using the detection method of claim 7, and the minimum template copy number that the kit can detect is analyzed; (3) Results: The test results show that the detection limit of the kit is 1 copy / uL and the total detection time is 35 min. It can be seen that the test kit is fast and highly sensitive, and can play an important role in rapid emergency detection.

9. The detection method according to claim 7, characterized in that: The detection method includes repeatability analysis, and the repeatability analysis method is as follows: (1) Sample processing: The viral RNA transcribed in vitro was used as the template for detection at a concentration of 1 copy / uL; (2) Detection: The reaction solution was prepared using the detection method described in claim 7, and the detection was repeated 10 times. Then, the reaction tube was placed on a fluorescence PCR instrument for detection. After the reaction was completed, the fluorescence curve was observed, and the repeatability of the detection by the kit described in this invention was analyzed. (3) Results: The test results showed that the kit was positive 10 times at a concentration of 1 copies / uL, which met the expected results; it can be seen that the test kit has good repeatability.

10. The detection method according to claim 7, characterized in that: The detection method includes specificity analysis. The method for the specificity analysis is as follows: (1) Sample processing: Select a group of clinically positive samples of respiratory disease-related pathogens, including influenza A H1N1 virus, influenza A H3N2 virus, influenza B virus, SARS-CoV-2, respiratory adenovirus, respiratory syncytial virus, rhinovirus, Haemophilus influenzae, Legionella, Streptococcus pneumoniae, Mycoplasma pneumoniae, monkeypox virus, plus in vitro transcribed Nipah virus RNA (10^5 copies / uL). (2) Detection: Using the detection kit, the above 13 samples were tested using the method of claim 7, and it was observed whether the kit would produce non-specific detection results; (3) Results: Based on the analysis of the fluorescence spectrum amplified by the detection kit, the kit was positive only for samples containing Nipah virus RNA, while the detection of the other 12 pathogens and the negative control was negative, proving that the method has good specificity.

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