WO2012122732A1 - Control/standard for nucleic acid detection of enveloped rna virus and applications thereof - Google Patents

Abstract

The present invention discloses a control of nucleic acid detection of enveloped RNA virus. The control is a simulated virus of enveloped RNA virus, and it has phospholipid bilayers and exogenous target RNA sequences. The viruses which are simulated include but are not limited to HIV, HCV, influenza virus, SARS-CoV.

Description

 Envelope RNA virus nucleic acid detection reference / standard and its application

 The invention belongs to the field of biotechnological inventions. The invention particularly relates to an enveloped RA virus nucleic acid detection reference/standard, which is mainly co-transformed by a vector carrying an exogenous sequence of interest and a vector containing an MLV gag-pol element, including or not including a viral membrane protein. Produced by a cell line produced by reference, is a pseudovirus with enveloped RA virus, mimics the phospholipid bilayer on the surface of the virus or further mimics the surface activity of the viral membrane protein, and has a specific enveloped RA virus structure. And physicochemical characteristics, no replication, no contagious. The coated RA virus nucleic acid detection reference product/standard product is packaged from eukaryotic cells by recombinant virus technology, and the reference product/standard sample particle can carry the exogenous purpose RA sequence through the genetic manipulation technology, and the reference product/standard product has The same viral envelope protein and similar viral structural characteristics and topological characteristics as the target virus can truly reflect the influence of various physicochemical environmental factors on the target virus. Background technique

 Nucleic acid detection technology, mainly including polymerase chain reaction (PCR) and nucleic acid hybridization technology, plays a huge role in the diagnosis of viruses because of its sensitivity, specificity, ease of operation, rapidity, high efficiency and high throughput. Become the first-line method of infectious disease detection. Nucleic acid detection technology is an effective diagnostic technique for early viral infection by qualitatively quantifying viral nucleic acids. For chronic viral infections, nucleic acid detection quantitative viral nucleic acid is the main method for evaluating viral replication and antiviral therapeutic effects. For the detection and diagnosis of RA virus, based on the polymerase chain reaction technology, the reverse transcription step (RT) is added, and the viral RNA is transcribed into cDNA by reverse transcriptase, and then amplified, which is the reverse transcription polymerase chain. Reactive reaction technique (RT-PCR).

 RT-PCR detection of RNA viruses mainly include: 1. Sample collection and storage; 2. Sample processing to extract nucleic acids; 3. Reverse transcription of R A into cDNA; 4. Amplification of cDNA template; 5. Detection and amplification. Each of the steps described above affects the final test results. In these processes, there are various physicochemical environmental factors, which may be caused by unsatisfactory sampling, residues of chemical reagents such as phenols, salts and ethanol in nucleic acid eluents during nucleic acid extraction, and may also originate from The intrinsic factors of the sample itself, such as heme, leukocyte DNA, heparin, bile and antibodies. In addition, the degradation of the target viral nucleic acid caused by R A enzyme and temperature in the storage and transportation environment cannot be ignored. Therefore, in order to reflect the reliability of nucleic acid detection results and to eliminate false negative results caused by various physicochemical environmental factors, setting reference products (including internal reference products and positive reference products) in samples is an indispensable part of qualitative and quantitative detection of nucleic acids.

The RA virus is further divided into non-enveloped RA virus and enveloped RA virus, but currently there are only two reference products for RA virus detection: (1) naked RA, (2) virus-like particles. The naked RA reference product is mainly derived from in vitro transcription, and the detection target is inserted into a plasmid carrying an in vitro transcriptase promoter, and a fragment synthesized by an in vitro transcriptase is provided with a detection target. RNA, then DNase is used to remove the DNA template from the RNA. But RNA itself is unstable and easy to be in the environment.

The R A enzyme is digested and degraded and cannot be stored for a long time, is inconvenient to transport, and cannot be compared with the R A in the virus. The most representative of the virus particle type reference product is "Armored RNA", which was developed by Ambion in 1996. "Armored RNA" uses the MS2 phage vector to insert the detection target into the MS2 phage packaging plasmid, and transforms the packaging plasmid into E. coli culture. The lysed bacteria are then purified by extraction with an organic solvent to obtain MS2 phage bearing the detection target. "Armored RNA" is easy to manufacture, simple to extract and purify, stable at room temperature or 4 °C for long-term storage, and has no infectivity. RA is protected by phage capsid protein. From the biological structure of virus, phage virus particle is a kind. The dense structure of the icosahedron, which is resistant to nucleases and various physicochemical environmental factors, is a choice as a standard or a positive reference.

 However, for different types of viral pathogens, differences in their biophysiological structural properties result in differences in resistance to the outside world. Especially for the enveloped RA virus, because the enveloped virus is enveloped by a lipid bilayer, its RA is neither exposed nor protected by capsid protein, and it is topologically impossible to be exposed to RA or clothing. Shell virus phage mimics, and thus it is not appropriate to simply use naked RA or capsid virus phage as a reference in the detection of enveloped RA virus.

 Viral pathogens that are harmful to human life and health in encapsulated RA viruses, including human acquired immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus, severe acute respiratory syndrome coronavirus (SARS-CoV), etc. , are viral pathogens that require early diagnosis, early detection, timely treatment and detection of therapeutic effects. In order to ensure the credibility of the detection and diagnosis of these enveloped RA viruses, and considering the viral structural biological characteristics and viral topology characteristics of the enveloped RA virus, the naked RA reference material or the capsid virus is used after sample collection. Phage as an internal reference can not accurately reflect the changes of the sample in the process of processing and detection, to achieve the function of the internal reference product, simulate the coated RA virus sample, and it is difficult to identify the false negative result caused by the environmental and chemical factors. Therefore, the ideal reference product should have the biophysical structural characteristics consistent with the biological characteristics of the test object. Only the development of a nucleic acid detection reference product having the same biological characteristics as the coated R A virus can truly monitor the pathogen.

The research of viruses has been greatly developed in recent years, such as the reverse genetic recombination technology of avian influenza virus. In 2003, French scientists made HCV pseudoviruses with infectious and target organ recognition capabilities. These technologies not only mark human viral bioengineering technology. Into the "art" era, more importantly, people can use it to evaluate neutralizing antibodies, look for specific epitopes, screen for possible receptors, identify possible vaccine strains, and create targeted Drug carriers and diagnostic techniques, and the like. A pseudovirus generally refers to a nanoparticle having a structure or functional unit that resembles a corresponding virus, and loses replication function after manipulation by a human gene, thereby losing viral toxicity. For various applications of pseudoviruses, it can be seen that the pseudovirus can simulate the enveloped virus well, reflecting the biophysical and topological characteristics of the enveloped virus, and thus can simulate the enveloped virus to withstand various samples in the sample. Environmental and chemical factors and the impact of these factors on enveloped viruses. From the perspective of virus detection and diagnosis, the pseudovirus is developed into a reference product/standard for the detection of enveloped RA virus nucleic acid, compared to the use of naked nucleic acid. Or non-enveloped capsid virus phage, more able to reflect the enveloped virus and its real situation in the sample.

 The virus-like packaging technology currently mainly uses the gag-pol protein of retrovirus such as human immunodeficiency virus (HIV) and murine leukocyte virus (MLV), which can recognize the gene containing the sequence by recognizing and binding to a specific sequence. The fragment is packaged in the inside of the protein particle, and the endoplasmic reticulum membrane expressing the viral membrane protein is packaged into particles, which are secreted outside the cell through the endoplasmic reticulum, Golgi apparatus and the like to form an artificial pseudovirus. Artificial pseudo-like viruses were engineered based on viral recombination technology and gene manipulation, and reference products/standards for detection of enveloped R A virus nucleic acids were developed. The viral biological structure of the coated RA virus nucleic acid detection reference product/standard is only a lipid bilayer coating, and the internal is a non-dense structure of the Gag protein binding nucleic acid, and the biological structure of the virus is consistent with the biological characteristics of the detection object. Biophysiological structural characteristics and topological characteristics. In nucleic acid detection (including polymerase chain reaction PCR and nucleic acid hybridization), the ideal reference should also meet the process of simulating the target virus by nucleic acid detection technology, including the same specificity and sensitivity, and the target virus. Synchronization is detected to maximize the simulation of the target viral nucleic acid detection process.

 The invention develops a coated RA virus nucleic acid detection reference product/standard product, and uses the HCV virus nucleic acid diagnosis as an example to illustrate the application of the coated RA virus nucleic acid detection reference product/standard product, and achieves the following purposes: The biological characteristics and topological characteristics of the virus with the same biological characteristics of the test object can truly reflect the influence of various physicochemical environmental factors on the target virus; the reference product/standard nucleic acid is RA, and the target containing the target RNA virus detection is included. The sequence or unique artificial target sequence has the same specificity and sensitivity as the target virus detection, and is detected synchronously to maximize the simulation of the target viral nucleic acid detection process; the coated RA virus nucleic acid detection reference/standard can be used for nucleic acid detection technology. , including polymerase chain reaction (PCR) and nucleic acid hybridization techniques. Summary of the invention

 The present invention relates to an enveloped RA virus nucleic acid detection reference product/standard, which is co-transfected with a plasmid of a viral membrane protein by a vector containing a MLV gag-pol element and a reference vector carrying an exogenous target sequence. Produced as a cell line, it is a pseudovirus with enveloped RA virus. It can simulate the biological and topological characteristics and physicochemical characteristics of the specific enveloped RA virus. It has no replication ability and no infectiousness. The coated RA virus nucleic acid detection reference product/standard product is packaged from the eukaryotic cell by recombinant virus technology, and the reference product particle can carry the exogenous target RA sequence through the genetic manipulation technology, and the exogenous target RA sequence is carried through the genetic operation. Technology can be inserted or replaced.

In order to make the detection of the coated RA virus nucleic acid detection reference/standard have the same specificity and sensitivity as the target virus, the nucleic acid detection process of the target virus can be simulated to the maximum extent, and the reference product/standard particle carries the exogenous purpose RA inside. The sequence may include PCR-pair or pairs of detection primers for the target virus, one or more detection probes for the target virus or probes not related to the target virus, and the length of the reference/standard detection target and the target virus detection target. , the nucleotide component, and the dissolution temperature must be the same; the target specificity can also be detected by hybridization with the nucleic acid molecule either alone or simultaneously A target sequence that is consistent with sensitivity. When the enveloped RA virus nucleic acid detection reference is used as a positive reference, the internal RA sequence carried therein contains one or more segments of the entire detectable segment of the detection target or the target viral gene consistent with the detection of the target virus. When the coated RNA virus nucleic acid detection reference product is used as an internal reference product, the target RA sequence carried therein contains a quantitative PCR detection probe irrelevant to the target virus or a nucleic acid molecule containing hybridization detection target specificity and sensitivity but with the target virus Unrelated target sequences.

 Coated RA virus nucleic acid detection reference/standard can be used in enveloped RA viruses, including but not limited to: human acquired immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus, severe acute respiration Syndrome Coronavirus (SARS-CoV). Detailed description of the invention

 1. The upstream untranslated sequence of the retrovirus and the recognition sequence R-U5-PSI and the downstream non-translated sequence U3-R were separately cloned from the murine leukemia virus MLV, and a polyclonal cleavage site was added to the two cis elements. Point MCS, splicing and ligating to pCMV vector by Infusion technology, named pTAR, see Example 1 to facilitate insertion or replacement of the foreign vector sequence of the vector plasmid, and the maximum length of the inserted or substituted exogenous target sequence is at least 3000 bp. The exogenous gene of interest can be inserted or replaced and contained within the enveloped RA virus nucleic acid detection reference to establish a target vector.

 2. The reference insert/standard target vector pTAR provides a polyclonal insertion site MCS that can insert a target sequence of a plurality of viruses, and the target sequence can be a detection target sequence of a target virus or a chimeric sequence of a plurality of detection targets, or Multiple target viruses detect the chimeric sequence of the target. Example 2 Designing the detection target sequence of hepatitis C virus (HCV) and the chimeric sequence of the detection target sequence of human acquired immunodeficiency virus (HIV) by sequence alignment analysis, in the reference target vector pTAR polyclonal cleavage site The point MCS inserts the HCV and HIV internal reference target sequences for PCR detection to have the same specificity and sensitivity as HCV detection or HIV detection, and is simultaneously detected to maximize the HCV or HIV nucleic acid detection process. With the same specificity and sensitivity, the primer sequence contained in the target sequence of the internal reference is identical to the primer for detecting the virus, and the probe sequence contained in the target sequence of the internal reference is the same as the length of the probe sequence for detecting the viral nucleic acid. The base components are the same, the dissolution temperature is the same, but the specific sequence is different, and the detection report signal is different. The internal reference containing HCV and HIV chimeric targets was named pTAR-HCV/HIV.

The pTAR-HCV/HIV internal reference target plasmid, MLV gag-pol element plasmid was co-transfected into the reference production cell line 293T, and the HCV membrane protein granules or non-transfected membrane protein granules were transfected, and the surface was packaged with or without virus. HCV/HIV internal reference for membrane proteins. The HCV membrane protein HCV/HIV internal reference is named IC-HCV/HIV-1, and the surface does not carry the viral membrane protein. The HCV/HIV reference product of the cell phospholipid bilayer is named IC-HCV/HIV- O o has an enveloped RNA virus nucleic acid detection internal reference, which mimics the phospholipid bilayer on the surface of the virus or further mimics the viral membrane protein surface activity. Encapsulated RA virus nucleic acid test After the reference product is mixed with the virus sample, it can reflect the biophysiological degradation process of the target virus due to the same reason when it is in the same physicochemical environment as the target virus, thereby reflecting the reliability of the final nucleic acid detection result of the target virus.

 3. The target sequence of the HCV/HIV internal reference product has the same specificity and sensitivity. Specifically, the primer sequence contained in the target sequence of the internal reference product is identical to the primer for detecting the virus, and the target sequence contained in the internal reference product is included. The probe sequence has the same length as the probe sequence for detecting the viral nucleic acid, the base components are the same, and the dissolution temperature is the same, but the specific sequence is different. Through the specific HCV-positive reference product and the target sequence of HCV internal reference product RT-PCR amplification test, the amplification efficiency of the detection target in the internal reference product and the positive reference product is compared, and the verification theory design is basically consistent with the practice experiment, and the internal reference is verified. The rationality and feasibility of the product design.

4. In the nucleic acid detection of HCV, in addition to the detection target sensitivity of the HCV virus nucleic acid detection internal reference product and the HCV virus sample, the theoretical and actual detection are consistent with each other. In the PCR amplification process, the internal reference product and the viral nucleic acid compete. In the various components of the reaction system, the amount of the internal reference product may cause a deviation of the target virus detection signal, or the internal reference product may not reach the purpose of the internal reference. Therefore, it is necessary to rationally use the HCV virus nucleic acid reference product within a certain range. The results of Example 4 show that the internal reference product titer of 10 ⁴ or less does not affect the detection of the positive reference product, that is, it does not affect the detection of the virus sample, and when the internal reference product titer is above 10 ³ , the positive reference product is not dropped. The degree of influence is greatly deviated, that is, the internal reference product is not biased due to the content of the virus sample, so 10 ⁴ is the optimum titer of the internal reference product.

 5. The HCV/HIV internal reference product is a membrane-like pseudovirus, and its viral membrane protein theoretically has the same tolerance characteristics as the true virus. After mixing with the virus sample, it can reflect the target virus when it is in the same physicochemical environment as the target virus. Biophysiological degradation process caused by the same cause. Example 5 The correlation between the detection change of the internal reference nucleic acid and the detection change of the HCV viral nucleic acid in the HCV positive serum and under various conditions of the HCV envelope was evaluated in the HCV envelope, and the HCV envelope was reflected. The RA virus nucleic acid detection reference is related to the tolerance of HCV virus.

6. The role of HCV virus nucleic acid detection inside reference products, on the one hand, reflects the biophysical degradation process of HCV virus caused by various reasons, and evaluates the detection of HCV virus nucleic acid by detecting the detection efficiency and detection value of a certain amount of internal reference products. The reliability of the process; on the other hand, the HCV internal reference and virus have very close detection sensitivity, and the internal reference of the known titer can be used to relatively quantify the target virus. The HCV virus nucleic acid detection standard has the same detection target and detection signal as the virus, and can be used as a HCV virus nucleic acid detection standard for drawing a standard curve in the nucleic acid detection process. Since the HCV virus nucleic acid detection positive reference product is a pseudo-RA virus, its nucleic acid is RA. Compared with the past plasmid as a standard curve, the HCV virus nucleic acid detection positive reference nucleic acid is consistent with the sample nucleic acid property, and is simultaneously reversed during the detection process. Recording, consistent in reverse transcription efficiency, is therefore more informative in applications such as quantification or comparison, and more reflective of the effects of human or systematic errors in the detection process. DRAWINGS 1/8: Target vector pTAR map

 2/8: HCV/HIV detection target construction map

 3/8: HCV/HIV detection target construction identification map

 4/8: Compared with the amplification efficiency of HCV-PC and HCV-IC, there is a good linear relationship between the detected Ct of HCV reference and HCV positive reference and the negative logarithm of dilution. The similar slope reflects the similar detection. Sensitivity, by calculating amplification efficiency

=10 (1/slope) -1, the amplification efficiencies of the HCV internal reference and the HCV positive reference are 1.07 and 1.01, respectively.

5/8: Exploratory usage and detection values in the total detection of HCV virus nucleic acid detection internal reference products and HCV positive reference products. The internal reference product titer of 10 ⁴ or less does not affect the detection of the positive reference product, that is, it does not affect the detection of the virus sample, and when the internal reference product titer is above 10 ³ , it is not affected by the titer of the positive reference product. A large deviation occurs, that is, the internal reference product is not biased due to the virus sample content, so 10 ⁴ is the optimum titer of the internal reference product.

 6/8: Correlation between the change in the detection of the internal reference nucleic acid and the detection of the HCV viral nucleic acid in the HCV-positive serum and under various conditions of the HCV envelope. In a normal temperature environment, the presence of nucleases or other unknown factors in the sample may cause degradation of the virus and its nucleic acid in the sample, resulting in a change in the amount of detection. The lower the temperature, the better the sample is preserved.

 7/8: The amount of HCV viral nucleic acid reference was stable in multiple serum samples.

 8/8: HCV virus nucleic acid detection positive reference / standard used to draw a standard curve during nucleic acid detection. Detailed ways

 The following examples are illustrative of the invention, but are not intended to limit the scope of the invention. Example 1 Construction of reference target vector

 The reference target vector is based on the reverse transcription mechanism of retrovirus, and the upstream non-translated sequence of the retrovirus and the recognition sequence R-U5-PSI and the downstream non-translated sequence U3-R are separately cloned from the murine leukemia virus MLV, and Polyclonal cleavage site MCS was added to the two cis-elements, and spliced to the pCMV vector by Inflision technique, designated as pTAR, and the pTAR structure map is shown in Fig. 1. Design two pairs of R-U5-PSI and U3-R primers according to Inflision technical requirements:

TCCAACCCCCAAAACACTATGATTT-3 '

U3-Rf: 5'-GTCGCGAGGCCTGTCGACAATGAAAGACCCCACCAAATTG-3 ' The nucleic acid of murine leukemia virus MLV was extracted using QIAGEN's QIAamp Ultraseus virus kit. Specific method: Take 1ml virus sample to 2ml EP tube and balance the temperature to 15 °C -25 °C, add 800ul Buffer AC, add 5.6ul carrier RNA to the lid of EP tube, then vortex for 10s to mix; incubate for 10 minutes at room temperature, Centrifuge at 3200 rpm for 3 minutes, discard all supernatants; add 300 ul Buffer AR (60 °C preheat) plus 20 ul of proteinase K, vortex to completely dissolve the precipitate; vortex for 5 seconds during a 10 °C water bath for 10 minutes, All were transferred to QIAamp Spin Column 6000 rpm for 1 minute to discard the waste liquid; force B 500ul Buffer AWl 7500 rpm centrifuge for 1 minute to discard the waste liquid, force B 500ul Buffer AW2 15000 rpm centrifuge for 3 minutes to discard the waste liquid, force B 30ul Buffer AVE 7500rpm centrifuge 1 In minutes, collect the RA-containing eluate and store at -20 °C.

Reverse transcription of extracted virus RA using Invitrogen Reverse Transcription Kit (Super Script II), specific method: Take 1.5ml centrifuge tube, add deionized water 5ul, random primer (lOuM) luK sample RA 5ul, dNTP mix ( 10mM Lul, incubate at 65 °C for 5 minutes, place on ice, force B 5 X First-strand Buffer 4ul, 0.1M, DTT 2uK RNaseOUT lul, incubate for 2 minutes at 25 °C, add SuperscriptTM II RT lul, 25 °C Incubate for 10 minutes, incubate at 42 °C for 50 minutes, incubate at 70 °C for 15 minutes, and take 2 ul of reverse transcript for PCR amplification. Two pairs of R-U5-PSI and U3-R primers were used to obtain two fragments of R-U5-PSI and U3-R. PCR reaction system: lOxHigh Fidelity PCR Buffer 5 ul; lOmM dNTP mixture lul; 50 mM MgS0 ₄ 2 ul; Primer lOuM Forward/Reward lul; Platinum Taq High Fidelity 0.5 ul (2.5 unit); water 37.5 ul; The total reaction volume is 50 ul. Reaction conditions: 94 ° C for 2 minutes; 94 . C 30 seconds, 65 V 40 seconds, 72 °C 1 minute, 30 cycles; 72 °C 3 minutes.

 The PCR products of the pCMV vector, R-U5-PSI and U3-R were linearized with Sac I and subjected to 1% agarose electrophoresis (TBE electrophoresis buffer: Tris 108 g, Na2EDTA*2H20 7.44 g, boric acid 55 g) , dilute to a volume of 1 L of deionized water; cut off the target fragment and the carrier into a weighed 1.5 ml EP tube and purify with QIAEX II Gel Extraction Kit: Add 3 volumes of Buffer QX1 (100 mg plus 300 ul QX1). Force B 30ul Buffer QIAEX II Then 50 ° C water bath for 10 minutes, during which the vortex oscillates every two minutes. Centrifuge at 13000 rpm for 30 seconds, discard the supernatant, add 500 ul of QIAEX I once, wash twice with Buffer PE 500 ul, discard the supernatant, and air dry for 15 minutes until the precipitate turns white. Incubate with 20 ul TE vortex for 30 minutes, incubate for 5 minutes at 13,000 rpm, centrifuge for 30 seconds, transfer the supernatant to a 1.5 ml EP tube, and measure the concentration.

According to the Infusion technology principle, the linearized pCMV vector and the R-U5-PSI fragment have 15 to 25 bases, and the R-U5-PSI fragment and the U3-R fragment also have 15 to 25 bases. The U3-R fragment and The linearized pCMV vector also has a 15 to 25 base overlap, and the Infusion technique allows it to be spliced according to the coincident portion. Clonetech's Inflision kit was used as follows: 5 In-Fusion buffer 2ul, Enzyme lul, linearized CMV vector, R-U5-PSI fragment, U3-R fragment, water, total volume 10ul. The amount of the carrier and each fragment is such that the molar ratio thereof is 2:1. The Infusion mixture was treated at 37 °C for 15 minutes, at 50 °C for 15 minutes, 40 ul of TE buffer was added to make up 50 ul, and 2.5 ul was converted to DH5a. The transformed DH5ct was placed in ice for 30 minutes, heated at 42 ° C for 45 seconds, and then placed in ice for 1 minute. LB medium was added, and cultured at 37 ° C for 60 minutes with shaking. LOOul was coated with ampicillin-containing L-agar plate medium, and cultured at 37 ° C for 16 hours to form a single colony. The single colony was cultured for 8 hours in ampicillin-containing LB medium, and the culture solution was cultured for 8 hours with QIAprep Spin Miniprep. Kit small plasmid: Pour the bacterial solution into the labeled 1.5ml centrifuge tube, 13000rpm, 4 ° C, 3 minutes, centrifuge to remove the supernatant; force B 250ul Buffer Pl, mix, force B 250ul Buffer P2, quickly Gently invert 4-6 times; add 350ul Buffer N3, invert 4-6 times to mix, white floc is precipitated, 13000rpm, 4°C, 10 minutes; Put QIAprep spin column in 1.5ml EP tube (collect Waste liquid) Then transfer the supernatant into the QIAprep spin column, 8000 rpm 4 ° C for 1 minute; discard the waste liquid, add 750 ul Buffer PE, 13000 rpm, 4 ° C, 1 minute, discard the waste liquid, 13000 rpm, 4 ° C, After centrifugation for 1 minute, the QIAprep spin column was transferred to a new 1.5 ml centrifuge tube, and 30 ul of Buffer EB was added vertically to the membrane. After standing for 1 minute, the liquid monoclonal plasmid was obtained by centrifugation at 4 ° C, 13000 rpm, 1 min. Sequencing was identified correctly. See Figure 1. Example 2 Application of pTAR and establishment of HCV/HIV reference products

 The detection target sequence of hepatitis C virus (HCV) and the detection target sequence of human acquired immunodeficiency virus (HIV) were designed by sequence alignment analysis, and the target sequences of the two viruses were spliced into a chimeric sequence in the chimeric sequence. The target sequence is not limited to the above two viruses. The reference target vector pTAR polyclonal cleavage site MCS was inserted into the HIV and HCV internal reference target sequence for PCR detection, and was named as the probe sequence of the upstream and downstream, including the primer sequence.

 HCV internal reference target sequence HCV-IC

 HCV-F: 5 '-AGTAGYGTTGGGTYGCGAAAG-3 '

 HCV-R: 5 '-GAGACCTCCCGGGGCACTC-3 '

 HCV-IC Probe: HEX-ACACCATACGTACCAGCGTAG-ECLIPSE

 HIV reference product target sequence HIV-IC

 HIV-F: 5 '-CAGGTCTTCCCGACGATGAC-3 '

 HIV-R: 5 '-ACTTGACTGGCGACGTAATCC-3 '

 HIV-IC Probe: 5'- HEX-TGAACTTCCCGCCGCCGTTGTTGT-ECLIPSE-3'

 The gene synthesizes the target sequence of the above primer probe and will be installed on the TA clone for the next genetic manipulation.

 1. Application of pTAR: Construction of pTAR-HCV/HIV-IC

The target TA clones obtained by the synthesis were digested with EcoRI/Sal l, respectively, and the vector was digested with the corresponding enzyme, and ligated to the reference target vector pTAR using T4 ligase. The specific operation is as follows: The TA clone of the synthetic target sequence and the reference target vector pTAR were digested with the above restriction endonuclease, and the digestion system was: 10xNEBuffer3 10ul, restriction endonuclease Each enzyme was 5 ul, BSA lul, plasmid DNA 5 ug, double distilled water to make up 100 ul, and incubated at 37 ° C for 2 hours. 1% agarose electrophoresis (TBE electrophoresis buffer: Tris 108 g, Na2EDTA*2H20 7.44 g, boric acid 55 g, deionized water to 1 L); cut off the target fragment and carrier to a weighed 1.5 ml EP tube Purification with QIAEX II Gel Extraction Kit: Add 3 volumes of Buffer QXl (100 mg plus 300ul QXl). Force B 30ul Buffer QIAEX II Then water bath at 50 ° C for 10 minutes, during which the vortex oscillates every two minutes. Centrifuge at 13000 rpm for 1 minute, discard the supernatant, add 500 ul of QIAEX I once, wash twice with Buffer PE 500 ul, discard the supernatant, and air dry for 15 min until the precipitate turns white. The mixture was shaken for 30 seconds by adding 20 ul of TE buffer, placed for 5 minutes, centrifuged at 13,000 rpm for 30 seconds, and the supernatant was transferred to a 1.5 ml centrifuge tube to measure the concentration. After calculation, the target gene fragment and the pTAR vector gene fragment were ligated in a 3:1 molar ratio with T4 ligase (Invirtogen), and the ligation system: 5X Ligase Reaction Buffer 4ul, pTAR vector 30finol, target gene fragment 90fmol, T4 DNA Ligase 0.1 U, deionized water was added to 20 ul, and the mixture was allowed to stand at room temperature for 1 hour. 2ul was converted to DH5a, placed in ice for 30 minutes, heated at 42 °C for 45 seconds, and then placed in ice for 1 minute. LB medium was added, and the mixture was incubated at 37 ° C for 60 minutes with shaking. LOOul was applied to an L-agar plate medium containing ampicillin, and cultured at 37 ° C for 16 hours to form a single colony, and the single colony was cultured for 8 hours in an ampicillin-containing LB medium. The bacterial culture solution was cultured for 8 hours. The plasmid was extracted with QIAprep Spin Miniprep Kit: The bacterial solution was poured into a labeled 1.5 ml centrifuge tube, centrifuged at 13,000 rpm, 4 ° C for 3 minutes, and the supernatant was discarded by centrifugation; 250 ul Buffer PI was added. Mix well (without blocky precipitation), add 250ul Buffer P2, quickly invert 4-6 times; add 350ul Buffer N3, invert 4-6 times to mix, white floc is precipitated, 13000rpm, 4°C, 10 minutes; place the QIAprep spin column in a 1.5ml EP tube (collect the waste) and then transfer the supernatant to the QIAprep spin column, centrifuge at 8000rpm for 4 minutes at 4°C, discard the waste; force B 750ul Buffer PE, 13000rpm , centrifuge at 1 °C for 1 minute, discard the waste solution; centrifuge at 13000 rpm, 4 °C for 1 minute, transfer the QIAprep spin column into a new 1.5 ml centrifuge tube, add 30 ul of Buffer EB vertically to the membrane, and place for 1 minute. Thereafter, a liquid monoclonal plasmid was obtained by centrifugation at 13,000 rpm for 1 minute. The pTAR-HCV/HIV-IC sequencing was correctly identified. See Figure 2

 2. Establishment of HCV/HIV reference products

The pTAR-HCV/HIV-IC internal reference target plasmid and the MLV gag-pol element plasmid were co-transfected into the reference production cell line 293T, and the HCV membrane protein granules or non-transfected membrane protein granules were transfected, and packaged with or without An HCV/HIV reference product carrying a viral surface membrane protein. The surface carrying HCV membrane protein HCV/HIV internal reference product named IC-HCV/HIV-1, the surface does not carry viral membrane protein, only the HCV/HIV reference material of the cell phospholipid bilayer is named IC-HCV/HIV- 0. The specific operations are as follows: pTAR-HCV/HIV-IC internal reference target plasmid, MLV gag-pol element plasmid, HCV membrane protein particles were adjusted to 0.1 ug/ul by UV spectrophotometer, and the three plasmids were co-transfected. The reference product production cell line 293T, the surface of the packaging carries the HCV membrane protein HCV/HIV internal reference, named IC-HCV/HIV-1; and in the case of transfection without the addition of HCV membrane protein particles, only pTAR-HCV/HIV - IC internal reference target plasmid, MLV gag-pol element plasmid, two plasmids co-transfected reference production cell line 293T, packaging surface does not carry viral membrane protein, only cell phospholipid bilayer The HCV/HIV internal reference was named IC-HCV/HIV-0. Using the PolyFect Transfection Kit, the pTAR-HCV/HIV internal reference target plasmid, the MLV gag-pol element plasmid (O.lug/ul), each 8 ul, and the HCV membrane protein granule (O.lug/ul) 3 ul were mixed; pTAR- The HCV/HIV internal reference target plasmid and the MLV gag-pol element plasmid (O.lug/ul) were mixed 8 ul each, and then mixed with DMEM 80 ul, mixed with 20 ul of transfection reagent, and allowed to stand at room temperature for 5 to 10 minutes. 293T cells were washed twice with 0.01M PBS, digested with 0.25% Trypsin EDTA, and mixed with complete medium, dispersed, counted, 1.5 million cells per well of 6-well plate, 1.5 ml of final volume per well, to be transfected into plasmid After dilution with 600 ul of complete medium, a 6-well plate was added and patted to mix the plasmid complex in the cell suspension. Incubate for 6 hours at 37 ° C in a 5% C02 carbon dioxide incubator, aspirate the transfection mixture, gently wash the cells 3 times with PBS, and add the complete medium for further 72 hours. The supernatant was collected, filtered through a 0.45 um PVDF membrane, and stored at 4 ° C, containing the supernatant of the HCV/HIV reference.

 Verification of HCV/HIV reference products, specific methods: The HCV/HIV reference nucleic acid was extracted using QIAGEN's QIAamp Ultraseus virus kit. The extraction procedure is described in Example 1. HCV-F, HIV-R primer RT-PCR amplification of HCV/HIV reference nucleic acid, using One step primescr-iptTM RT-PCR Kit (TaKaRa) RT-PCR reaction system: 2xOne Step RT-PCR Buffer III 10ul , TaKaRa Ex TaqTM HS ( 5 U/ul) 0.5ul, PrimeS- criptTM RT Enzyme Mix II 0.5uK upstream primer final concentration 200nM, downstream primer final concentration 200nM, sample nucleic acid 2ul, no RA enzyme deionized water to make up 20ul. RT-PCR reaction conditions: reverse transcription 42 ° C 5 minutes reverse transcriptase thermal variability inactivation 94 ° C 10 s; 95 ° C denaturation 5 s, 60 ° C annealing extension 30 s, a total of 30 cycles. The RT-PCR product was verified by electrophoresis for fragment size, see Figure 3. Example 3 Detection sensitivity of the internal reference product and HCV virus of R A virus nucleic acid in HCV envelope

 1. Establishment of HCV detection positive reference

 HCV positive reference target sequence HCV-PC

 HCV-F: 5 '-AGTAGYGTTGGGTYGCGAAAG-3 '

 HCV-R: 5 '-GAGACCTCCCGGGGCACTC-3 '

 HCV-PC probe: 5'- FAM-AAGCACCCTATCAGGCAGTAC-ECLIPSE-3'

 The gene synthesizes the target sequence of the above primer probe and will be mounted on a TA clone for the next genetic manipulation, which contains a primer probe for detecting the HCV virus.

 Using the method of constructing pTAR-HCV/HIV-IC in Example 2, the sequence of HCV-PC was inserted into the vector pTAR to obtain the HCV-positive reference target vector pTAR-HCV-PC.

An HCV-positive reference was established using the method of establishing an HCV/HIV reference in Example 2.

共 4个稀释度。 使用 QIAGEN公 司 QIAamp Ultrasens virus 试剂盒提取核酸，提取步骤见实施例 1描述。使用 HCV-F, HCV-R 引物， HCV-PC 探针和 HCV-IC 探针对核酸样品进行实时荧光定量检测， 荧光定量 PCR设置 为 FAM与 HEX两种荧光检测，用 One step primescriptTM RT-PCR Kit (TaKaRa公司） RT-PCR 反应体系： 2xOne Step RT-PCR Buffer III lOuK TaKaRa Ex TaqTM HS ( 5 U/ul) 0.5ul、 PrimeScriptTM RT Enzyme Mix II 0.5uK 上游引物终浓度 200nM、 下游引物终浓度 200nM、 HCV-PC 探针和 HCV-IC 探针 2.5nM样品核酸 2ul、 无 R A酶去离子水补足 20ul。 RT-PCR 反应条件：反转录 42°C 5分钟反转录酶热变性失活 94 °C 10s; 95°C变性 5s， 60°C退火延伸 30s， 共 40个循环。 根据 PCR定量结果 Ct值绘制标准曲线与稀释度作相关性分析， HCV内参照 品与 HCV阳性参照品的检出 Ct与稀释度的负对数呈良好的线性关系， 相近的斜率反映了相 近的检测灵敏度，通过计算扩增效率 =10(1/斜率) -1， HCV内参照品与 HCV阳性参照品的扩增 效率分别为 1.07与 1.01， 见附图 4。 实施例 4 HCV病毒核酸检测内参照品在 HCV检测中最适使用量 2. Detection sensitivity of RAV nucleic acid detection internal reference product and HCV detection positive reference product of HCV envelope In order to maximize the simulation of target virus nucleic acid amplification process, detection of enveloped RA virus nucleic acid detection internal reference product The length of the needle is identical to the target virus detection probe, and the ratio of the four nucleotide residues of the detection probe is identical to the target virus detection probe, and the detection temperature (Tm value) of the detection probe is consistent with the target virus detection probe. In order to test the detection sensitivity of the HCV reference product detection target and the HCV virus detection target, the HCV internal reference product and the HCV positive reference product initial solution are mixed in equal volume, and then 10-fold serial dilution, respectively

A total of 4 dilutions. Nucleic acids were extracted using QIAGEN's QIAamp Ultrasens virus kit, and the extraction procedure is described in Example 1. Real-time fluorescence quantitative detection of nucleic acid samples using HCV-F, HCV-R primers, HCV-PC probes and HCV-IC probes, fluorescence quantitative PCR set to FAM and HEX fluorescence detection, using One step primescriptTM RT-PCR Kit (TaKaRa) RT-PCR reaction system: 2xOne Step RT-PCR Buffer III lOuK TaKaRa Ex TaqTM HS ( 5 U/ul) 0.5ul, PrimeScriptTM RT Enzyme Mix II 0.5uK upstream primer final concentration 200nM, downstream primer final concentration 200nM , HCV-PC probe and HCV-IC probe 2.5nM sample nucleic acid 2ul, no RA enzyme deionized water to make up 20ul. RT-PCR reaction conditions: reverse transcription 42 ° C 5 minutes reverse transcriptase thermal denaturation inactivation 94 ° C 10s; 95 ° C denaturation 5s, 60 ° C annealing extended 30s, a total of 40 cycles. Correlation analysis was carried out according to the Ct value of PCR quantitative results. The correlation between the detected Ct and the negative logarithm of the HCV positive reference and the HCV positive reference showed a good linear relationship. The similar slopes reflect similar The detection sensitivity was calculated by the amplification efficiency = 10 (1/slope) -1, and the amplification efficiencies of the HCV internal reference and the HCV positive reference were 1.07 and 1.01, respectively, as shown in Fig. 4. Example 4 HCV virus nucleic acid detection internal reference product is optimally used in HCV detection

In the nucleic acid detection of HCV, in addition to the detection target sensitivity of the HCV virus nucleic acid detection internal reference product and the HCV virus sample, the theoretical and actual detection are consistent with each other. In the PCR amplification process, the internal reference product and the viral nucleic acid compete for the reaction system. The various components, the internal reference product will cause the deviation of the target virus detection signal, or the internal reference product is insufficient to achieve the purpose of internal reference. Therefore, it is necessary to rationally use the HCV virus nucleic acid reference product within a certain range. In this protocol, HCV internal reference products and HCV positive reference products were used to simulate HCV virus, which were diluted 10 times and quantified as 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , 10 five titers, and the internal reference products were positive. Five titer samples of the reference product were mixed to obtain 25 mixed samples and five reference positive reference products without mixed internal reference products as controls. After the sample is mixed, the nucleic acid is extracted as described in Example 1, and the nucleic acid sample is subjected to real-time fluorescence quantitative detection using HCV-F, HCV-R primer, HCV-PC probe and HCV-IC probe, and the fluorescent quantitative PCR is set to FAM and HEX fluorescence detection, using One step primescriptTM RT-PCR Kit (TaKaRa) RT-PCR reaction system: 2 x One Step RT-PCR Buffer ΠΙ lOuK TaKaRa Ex TaqTM HS (5 U/ul) 0.5ul, PrimeScriptTM RT Enzyme Mix II 0.5ul upstream primer 200nM, downstream primer final concentration 200nM, HCV-PC probe and HCV-IC probe 2.5nM sample nucleic acid 2ul, RNase-free deionized water to make up 20ul. RT-PCR reaction conditions: reverse transcription 42 ° C 5 minutes reverse transcriptase thermal denaturation inactivation 94 ° C 10s; 95 ° C denaturation 5s, 60 ° C annealing extended 30s, a total of 40 cycles. The results show that the internal reference product titer is 10 ⁴ Or the following does not affect the detection of positive reference products, that is, does not affect the detection of virus samples, and when the internal reference product titer is above 10 ³ , there will be no large deviation due to the influence of the positive reference product titer, that is, it will not Because of the virus sample content, there is a bias in the quantification of the internal reference, so 10 ⁴ is the optimum titer of the internal reference, see Figure 5. Example 5 Correlation between RAV nucleic acid detection reference of HCV envelope and HCV virus tolerance

 The HCV/HIV reference product is a membrane-like pseudovirus, which is a reference product for the detection of enveloped RA virus nucleic acid. The viral membrane protein has the same tolerance characteristics as the true virus in combination with the virus sample, and the target virus. When in the same physicochemical environment, it can reflect the biophysical degradation process of the target virus caused by the same reason. The HCV/HIV internal reference product established in Example 2 is a reference reagent for RA virus nucleic acid detection of HCV envelope, and contains an internal reference target of HCV/HIV. Therefore, the RAV nucleic acid detection internal reference designed by the present scheme is HCV envelope. The correlation between the detection change of the internal reference nucleic acid and the detection change of the HCV viral nucleic acid in the HCV positive serum and under various conditions reflects the tolerance of the RAV nucleic acid detection reference product of the HCV envelope to the HCV virus.

The quantitative detection of the RAV nucleic acid detection reference of the HCV envelope was adjusted to a starting amount of 1.1 million copies/ml, and was adjusted and mixed with the HCV positive serum quantified by the viral load in a serum volume of 1 ml, and the serum ratio was greater than 90%, wherein The viral nucleic acid was approximately 1.6 million copies/ml (quantitative to the plasmid standard curve). The samples were mixed to prepare 40 parts, respectively a first group of: 10 parts by placing the room temperature environment; Second group: 4 ^Q C is placed 10 parts of the environment; third group: -40 ^Q C is placed 10 parts of the environment; -40 fourth group 10 parts placed in the ^Q C environment but repeatedly frozen and thawed. The starting time point is 0, and then the detection time points are 6 hours, 24 hours (first day), 2nd day, 3rd day, 7th day, 14th day, 21st day, 28th day, 42nd Day, day 56. One sample was taken from each experiment, and the fourth group of samples was completely frozen and thawed each time. In a normal temperature environment, the presence of nucleases or other unknown factors in the sample may cause degradation of the virus and its nucleic acid in the sample, resulting in a change in the amount of detection. The lower the temperature, the more favorable the sample is to preserve, see Figure 6. Nucleic Acid Extraction Procedure: HCV/HIV reference nucleic acid was extracted using QIAGEN's QIAamp Ultraseus virus kit. The extraction procedure is described in Example 1. Simultaneous detection of viral nucleic acids and internal reference products are performed on the extracted nucleic acids. The viral nucleic acid detection is the same as the primer used for the internal reference detection, the probe is different and the fluorescent signal carried by the probe is different. Fluorescence quantitative RT-PCR was set up for FAM and HEX fluorescence detection, HCV-F, HCV-R primers were used to reverse transcribe and amplify sample nucleic acid, using One step primescriptTM RT-PCR Kit (TaKaRa) RT-PCR reaction system : 2xOne Step RT-PCR Buffer III 10ul, TaKaRa Ex TaqTM HS ( 5 U/ul) 0.5ul, PrimeScriptTM RT Enzyme Mix II 0.5uK Rox Correction Dye 0.4ul, Virus Detection Probe 2.5nM 2ul, Internal Reference Detection Probe 2.5nM 2ul, the upstream primer has a final concentration of 200nM, the downstream primer has a final concentration of 200nM, the sample nucleic acid is 2ul, and the RA enzyme deionized water is used to make up 20ul. RT-PCR reaction conditions: reverse transcription 42 ° C 5 minutes reverse transcriptase thermal denaturation inactivation 94 ° C 10s; 95 °C denaturation 5s, 60 °C annealing extension 60s, a total of 40 cycles. Example 6 Application of HCV Virus Nucleic Acid Detection Reference in HCV Virus Nucleic Acid Detection

1. Application of HCV virus nucleic acid detection internal reference in HCV virus nucleic acid detection

 The role of HCV virus nucleic acid detection in internal reference products, on the one hand, reflects the biophysical degradation process of HCV virus caused by various reasons, and evaluates the detection process of HCV virus nucleic acid by detecting the detection efficiency and detection value of a certain amount of internal reference products. reliability. A HCV viral nucleic acid detection internal reference having a quantitative amount of about 110,000 copies/ml was mixed with 14 HCV serum samples, and the nucleic acid extraction process and the RT-PCR detection process were simultaneously carried out in Example 5. The HCV viral nucleic acid reference is stable in multiple serum samples and can be used as a basis for relative quantification, see Figure 7.

On the other hand, the internal reference and the virus have very close detection sensitivities, and the internal reference of the known titer can be used to relatively quantify the target virus. The specific method is as follows: 10 ⁴ copy number of HCV virus nucleic acid detection internal reference product and three titer HCV in vitro transcription RA mixed, HCV in vitro transcription RA copy number by spectrophotometric detection and theoretical calculation is 4.8 X 10 ⁴ , 4.8 X 10 ³ , 4.8 X 10 ² , by calculating the relative quantitative RA copy number of the HCV virus nucleic acid reference and the HCV RA detection Ct value, the experimental relative quantitative HCV RA copy number is in agreement with the theoretical calculation value, the result See Table 1. Specific methods: Real-time quantitative RT-PCR simultaneous detection of HCV internal reference products and HCV in vitro transcription of RA, fluorescence quantitative RT-PCR set to FAM and HEX two fluorescence detection, using HCV-F, HCV-R primer reverse transcription And amplification of sample nucleic acids, using One step primescriptTM RT-PCR Kit (TaKaRa) RT-PCR reaction system: 2xOne Step RT-PCR Buffer III lOuK TaKaRa Ex TaqTM HS ( 5 U/ul ) 0.5ul, PrimeScriptTM RT Enzyme Mix II 0.5uK Rox calibration dye 0.4ul, virus detection probe 2.5nM 2ul, internal reference detection probe 2.5nM 2ul, upstream primer final concentration 200nM, downstream primer final concentration 200nM, sample nucleic acid 2ul, no RA enzyme deionized water to make up 20ul . RT-PCR reaction conditions: reverse transcription 42 ° C 5 minutes reverse transcriptase thermal denaturation inactivation 94 ° C 10s; 95 ° C denaturation 5s, 60 ° C annealing extension 60s, a total of 40 cycles. The results showed that the reliability of HCV virus nucleic acid detection reference HCV virus nucleic acid detection process can also be applied to HCV viral load detection.

 RNA Ct value theory RNA copy number IC Ct value IC copy number Relative quantification RNA copy number

 25.005 4.80E+04 27.295 1.E+04 49424.17104

 29.055 4.80E+03 27.375 1.E+04 3096.770997

 34.185 4.80E+02 27.33 1.E+04 83.70034975

2. HCV virus nucleic acid detection positive reference / standard application in HCV virus nucleic acid detection.

The HCV virus nucleic acid detection positive reference product/standard product is a reference product with the same detection target and detection signal as the virus, and can be used as a HCV virus nucleic acid detection standard for drawing a standard curve in the nucleic acid detection process. Since the HCV virus nucleic acid detection positive reference is a pseudo-RA virus, its nucleic acid is RA, compared with the past plasmid as a standard curve. HCV virus nucleic acid detection positive reference nucleic acid is consistent with the nature of the sample nucleic acid. Simultaneous reverse transcription during the detection process is consistent in reverse transcription efficiency. Therefore, it is more useful in quantitative or comparative applications, and more reflective of the detection process. Or systemic error effects. A known amount of HCV virus nucleic acid detection positive reference nucleic acid was extracted, the nucleic acid was serially diluted 10 times to prepare a standard curve, and HCV virus detection primers and probes were used, and One step primescriptTM RT-PCR Kit (TaKaRa) RT-PCR reaction was used. System: 2xOne Step RT-PCR Buffer III lOuK TaKaRa Ex TaqTM HS ( 5 U/ul) 0.5ul, PrimeScriptTM RT Enzyme Mix II 0.5uK Rox Correction Dye 0.4ul, Virus Detection Probe 2.5nM 2ul, Internal Reference Detection Probe 2.5nM 2ul, the upstream primer has a final concentration of 200nM, the downstream primer has a final concentration of 200nM, the sample nucleic acid is 2ul, and the RA enzyme deionized water is used to make up 20ul. RT-PCR reaction conditions: reverse transcription 42 ° C 5 minutes reverse transcriptase thermal denaturation inactivation 94 ° C 10s; 95 ° C denaturation 5s, 60 ° C annealing extension 60s, a total of 40 cycles. See Figure 8.

Claims

Claim  1. A coated RNA virus nucleic acid detection reference product, characterized in that the reference product is a pseudovirus resembling an enveloped RNA virus, which can simulate the structure and physicochemical characteristics of the enveloped RNA virus, and can truly react variously The influence of environmental and chemical factors on the target virus. No replication, no contagious.  The enveloped RNA virus nucleic acid detection reference according to claim 1, wherein the reference drug envelope is a phospholipid bilayer similar to the enveloped RNA virus, and may or may not express a specific enveloped RNA. The envelope protein of the virus.  3. The pseudo-virus of the specific enveloped RNA virus according to claim 1, the simulated virus includes but not limited to: human acquired immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV).  The enveloped RNA virus nucleic acid detection reference product according to claim 1, which is packaged from a eukaryotic cell by a recombinant virus technology, and allows the reference particle to carry an exogenous RNA of interest through a genetic manipulation technique. sequence.  5. The envelope protein with a specific enveloped RNA virus according to claim 2, including but not limited to: human acquired immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus, severe acute respiration Syndrome coronavirus (SARS-CoV) ₀  The enveloped RNA virus nucleic acid detection reference according to claim 1, which comprises an RNA target sequence for RNA virus nucleic acid detection.  The RNA target sequence for RNA viral nucleic acid detection according to claim 6, wherein the RNA target sequence is inserted or replaced by genetic manipulation.  The RNA target sequence for RNA virus nucleic acid detection according to claim 6, wherein the RNA target sequence can be a detection target sequence of a target virus or a chimeric sequence of a plurality of detection targets, or multiple targets. The chimeric sequence of the viral detection target.  The coated RNA virus nucleic acid detection reference product according to claim 1 and the RNA viral nucleic acid detection RNA target sequence according to claim 6, wherein the enveloped RNA virus nucleic acid detection reference product can accommodate The exogenous RNA target sequence is at least 3000 bp in length.  The RNA target sequence for RNA viral nucleic acid detection according to claim 6, which comprises a primer sequence for detecting a target virus.  The RNA target sequence for RNA viral nucleic acid detection according to claim 6, which comprises a specific detection probe sequence for detecting a target virus, or a detection probe sequence different from the target virus. The enveloped RNA virus nucleic acid detection reference according to claim 1, wherein the reference product and the target virus share the same detection primer, the same or different detection probe and the report signal during the detection. 13. The same detection probe and reporter signal according to claim 12, characterized in that the enveloped RNA virus nucleic acid detection reference is a positive reference for the detection of the target viral process.  14. The different detection probes and reporter signals according to claim 12, wherein the enveloped RNA virus nucleic acid detection reference is an internal reference for the detection of the target virus process.  The coated reference RNA virus nucleic acid detection positive reference according to claim 13, wherein the detection of the positive reference material of the enveloped RNA virus nucleic acid detection and the detection of the target virus are independent detection processes.  The enveloped RNA virus nucleic acid detection internal reference according to claim 14, wherein the detection of the enveloped RNA virus nucleic acid detection internal reference product is the same detection process as the detection of the target virus.  The enveloped RNA virus nucleic acid detection internal reference according to claim 14, wherein the detection of the internal reference product and the report signal for the detection of the target virus are independent of each other, and the detection of the target virus is not affected.  The enveloped RNA virus nucleic acid detection internal reference according to claim 14, wherein the length of the internal reference detection probe is identical to the target virus detection probe, and the target virus nucleic acid amplification process is simulated to the maximum extent.  The enveloped RNA virus nucleic acid detection internal reference according to claim 14, wherein the ratio of the four nucleotide residues of the internal reference detection probe is identical to the target virus detection probe, and the maximum Simulate the target virus nucleic acid amplification process.  The enveloped RNA virus nucleic acid detection internal reference according to claim 14, wherein the detection temperature of the internal reference detection probe (Tm value) is consistent with the target virus detection probe, and the target virus is simulated to the maximum extent. Nucleic acid amplification process.  21. The coated RNA viral nucleic acid detection reference according to claim 1, which can be used for qualitative detection of enveloped RNA viral nucleic acids, including but not limited to polymerase chain reaction (PCR) methods and nucleic acid molecule hybridization methods.  22. The coated RNA viral nucleic acid detection reference according to claim 1, which can be used for quantitative detection of enveloped RNA viral nucleic acids, including but not limited to polymerase chain reaction (PCR) methods and nucleic acid molecule hybridization methods.  23. The qualitative detection according to claim 21 and the quantitative detection according to claim 22, wherein the coated RNA virus nucleic acid detection internal reference product is added to the sample as soon as possible after sampling, and participates in various treatments of the sample. And the nucleic acid detection process.  24. The qualitative detection according to claim 21 and the quantitative detection according to claim 22, wherein the coated RNA virus nucleic acid detection positive reference product is not mixed with the sample, and participates in various treatments in parallel with the sample. Nucleic acid detection process.