CN113584228A - Quality control product for nucleic acid detection of seven RNA viruses and preparation method thereof - Google Patents

Abstract

The invention discloses a quality control product for nucleic acid detection of seven RNA viruses and a preparation method thereof, belonging to the field of clinical laboratory and applied molecular biology. The invention has a nucleotide sequence shown in a sequence table SEQ ID NO.3-SEQ ID NO. 13. The invention adopts an optimized MS2 phage virus-like particle packaging technology to respectively wrap the sequences of the recombinant conventional detection regions of seven RNA viruses into MS2 capsid protein, thereby preparing the MS2 virus-like particle nucleic acid detection quality control products of the seven viruses. The quality control product fully simulates the structure of natural virus particles, has stable performance, nuclease resistance and no any infection risk, and realizes the quality monitoring and evaluation of the whole process of nucleic acid extraction, reverse transcription and amplification detection. The quality control product has good universality and wide detection area, is suitable for the quality control of a conventional detection method and various detection kits, and overcomes the problem that different kits cannot share the quality control product.

Description

Quality control product for nucleic acid detection of seven RNA viruses and preparation method thereof

Technical Field

The invention relates to a quality control product for nucleic acid detection of seven RNA viruses and a preparation method thereof, belonging to the field of clinical laboratory and applied molecular biology.

Background

Acute respiratory infection often causes symptoms such as fever, cough, watery nasal discharge and the like, and some patients (such as children, patients with basic diseases and low immunity) often cause serious complications such as pneumonia, bronchitis and the like, and are the main causes of death of children within 5 years of age. Clinically, the types of pathogens causing acute Respiratory infection are many, and 80% of the pathogens are viral infections, and mainly include Influenza virus (IFV), Respiratory Syncytial Virus (RSV), Parainfluenza virus (PIV), rhinovirus (HRV), and the like. Coxsackievirus A16 type (Coxsackievirus A16, CA16) and Enterovirus71 type (Enterovirus71, EV71) are common important viruses causing hand-foot-and-mouth diseases of children, the hand-foot-and-mouth diseases caused by the Coxsackie virus A16 type and the Enterovirus71 type are difficult to distinguish clinically, and complications such as encephalitis, meningitis and the like can be caused to patients when the hand-foot-and-mouth diseases are serious. Because different pathogen infections have certain differences in the aspects of treatment modes, clinical prognosis and the like of patients, rapid and accurate identification of the pathogens has guiding significance for clinical early diagnosis and treatment, and can also effectively prevent further transmission of the pathogens in people.

At present, the molecular detection technology (such as real-time fluorescence reverse transcription PCR, multiplex PCR and the like) for carrying out amplification detection on a specific nucleic acid sequence of a virus genome has the advantages of rapidness, sensitivity and the like, greatly improves the sensitivity and specificity of virus detection, has excellent virus quantification and typing identification capabilities, and is the mainstream technology for laboratory virus detection at present. However, in the actual detection process, the sensitivity and specificity of the PCR method are very dependent on the efficiency of extraction of target nucleic acid from a sample, the specificity of primer and probe design, the rationality of enzyme amplification reaction condition design, and the like. Therefore, a quality control product having stable performance and definite characteristics is essential for quality control in nucleic acid detection.

At present, the nucleic acid detection quality control product of RNA virus mainly comes from plasmids, RNA transcribed in vitro, virus samples cultured by clinical specimens, lentivirus virus-like particles and MS2 virus-like particles. In a laboratory or a commercial kit, plasmid or in vitro transcribed RNA is usually used as a quality control material, however, the quality control material cannot simulate the process of nucleic acid extraction, cannot evaluate the extraction efficiency, cannot evaluate the reverse transcription process, and has the defects of instability, easy degradation and the like. Although virus samples cultured in clinical specimens can simulate the whole process from nucleic acid extraction to detection, they are dangerous for biological infection and are not easily available in large quantities. Virus-like particles from lentivirus packaging can evaluate the nucleic acid extraction process, further reducing the risk of viral infection, but also having the problems of low yield, high culture cost, potential infection risk, and the like. The MS2 virus-like particle prepared by adopting the MS2 phage virus-like particle packaging technology only consists of an MS2 virus protein capsid and an internally wrapped nucleic acid fragment, and the packaging of an exogenous RNA sequence can be realized by constructing a prokaryotic expression system. The MS2 virus-like particle has stable performance, the outer coat shell egg can protect the RNA wrapped inside from being degraded, can eliminate the risk of biological infection while simulating the whole detection process, is an ideal RNA virus simulation reference material, and is suitable for the preparation of the nucleic acid detection quality control products of common RNA viruses (such as respiratory syncytial virus, rhinovirus, parainfluenza virus, coxsackie virus A16, enterovirus71 and the like).

However, the virus-like particles prepared by the conventional packaging technology of the MS2 phage virus-like particles have the limitation on the size of the inserted exogenous sequence, and when the length of the particles exceeds 500bp, the packaging efficiency is rapidly reduced, the yield of the virus-like particles is greatly reduced, the product concentration is lower and is only about 10⁶copies/mL. Meanwhile, because of the difference of detection target areas of different kits, the application range of the existing quality control product is limited, and one quality control product can only be suitable for the corresponding detection kit, so that the quality control products of various kits are difficult to share at present. Therefore, the preparation of the safe and reliable RNA virus quality control product which can be expressed efficiently and has wide application range has important significance.

Disclosure of Invention

The first technical problem to be solved by the invention is to provide nucleic acid detection quality control products of seven common RNA viruses (including respiratory syncytial virus A, respiratory syncytial virus B, rhinovirus C, parainfluenza virus 1, parainfluenza virus 3, coxsackie virus A16 and enterovirus 71).

The second technical problem to be solved by the invention is to provide an efficient preparation method of the seven RNA virus nucleic acid detection quality control products.

In order to achieve the purpose, the invention adopts the following technical scheme:

seven RNA virus nucleic acid detection quality control products have nucleotide sequences shown in a sequence table SEQ ID NO.3-SEQ ID NO. 13;

(1) the RNA virus is respiratory syncytial virus A type virus and has nucleotide sequences shown in a sequence table SEQ ID NO.3 and a sequence table SEQ ID NO. 4;

(2) respiratory syncytial virus B type virus, having nucleotide sequences shown in sequence tables SEQ ID NO.5 and SEQ ID NO. 6;

(3) rhinovirus C type virus with nucleotide sequence shown in sequence table SEQ ID NO. 7;

(4) parainfluenza virus type 1 having a nucleotide sequence represented by SEQ ID NO.8 or SEQ ID NO.9 of the sequence Listing;

(5) parainfluenza virus type 3 having a nucleotide sequence represented by SEQ ID NO.10 or SEQ ID NO.11 of the sequence Listing;

(6) coxsackievirus A16 type virus, having a nucleotide sequence shown in a sequence table SEQ ID NO. 12;

(7) the enterovirus71 has a nucleotide sequence shown in a sequence table SEQ ID NO. 13.

The seven RNA virus nucleic acid detection quality control products are MS2 virus-like particle nucleic acid detection quality control products of seven RNA viruses.

The quality control product for nucleic acid detection is prepared by the following method:

1) constructing a double-expression system vector plasmid pACYC-MS2-virus simultaneously containing an MS2 bacteriophage gene sequence and a virus recombinant conventional detection region to obtain a recombinant vector;

2) transforming BL21(DE3) E.coli, IPTG induced expression and purifying MS2 virus-like particles by using sephadex gel filtration chromatography;

3) packaging the virus-like particles by MS2 and verifying the sequence correctness by adopting a series of methods of nuclease digestion, polyacrylamide gel electrophoresis, reverse transcription PCR, Sanger sequencing and real-time fluorescence reverse transcription PCR; absolute quantification is carried out by adopting liquid drop digital PCR;

4) diluting and subpackaging a sample by adopting DMEM diluent; lyophilizing with Christ ALPHA 1-2 type lyophilizer at 0.37mbar for 25 hr to obtain the final product.

The invention adopts the optimized MS2 phage virus-like particle packaging technology to respectively package the recombinant conventional detection areas of the seven RNA viruses, thereby obtaining the MS2 virus-like particle nucleic acid detection quality control product of the seven RNA viruses with high-efficiency expression and wide application range.

The seven common RNA virus nucleic acid detection quality control products provided by the invention have the advantages of particle structure similar to natural viruses, nuclease resistance, stable performance and high safety, so that the seven viruses can be conveniently monitored and evaluated from the whole process of nucleic acid extraction, reverse transcription and amplification detection; meanwhile, the quality control product can be suitable for the quality control of the conventional detection method and various detection kits so as to overcome the problem that different kits cannot share the quality control product.

In another aspect of the invention, a preparation method of the seven RNA virus nucleic acid detection quality control products is provided, which comprises the following steps:

1) constructing a double-expression system vector plasmid pACYC-MS2-virus simultaneously containing an MS2 bacteriophage gene sequence and a virus recombinant conventional detection region to obtain a recombinant vector;

2) transforming BL21(DE3) E.coli, IPTG induced expression and purifying MS2 virus-like particles by using sephadex gel filtration chromatography;

3) packaging the virus-like particles by MS2 and verifying the sequence correctness by adopting a series of methods of nuclease digestion, polyacrylamide gel electrophoresis, reverse transcription PCR, Sanger sequencing and real-time fluorescence reverse transcription PCR; absolute quantification is carried out by adopting liquid drop digital PCR;

4) diluting and subpackaging a sample by adopting DMEM diluent; lyophilizing with Christ ALPHA 1-2 type lyophilizer at 0.37mbar for 25 hr to obtain the final product.

The double expression system carrier plasmid pACYC-MS2-virus takes pACYCDuet-1 plasmid as a carrier, capsid protein gene and mature enzyme gene sequences of MS2 bacteriophage are respectively inserted into a multiple cloning site 1 of the plasmid in a restriction enzyme cutting mode, a recombinant conventional detection region sequence of related viruses is inserted into a multiple cloning site 2 of the plasmid, and the recombinant carrier plasmid can simultaneously express MS2 capsid protein and exogenous virus RNA.

The recombinant conventional detection region sequence covers the target region of most of the existing detection methods and detection kits, and the two ends of the recombinant sequence are connected with an optimized MS2 phage packaging signal and an optimized enzyme digestion site.

The real-time fluorescent reverse transcription PCR and liquid drop digital PCR quantitative method adopts a primer probe sequence which is a nucleotide sequence shown in a sequence table SEQ ID NO.14-SEQ ID NO. 46.

Specifically, the step 1 of constructing a dual expression system vector plasmid simultaneously containing an MS2 gene sequence and a virus recombinant conventional detection region refers to: inserting capsid protein gene and mature enzyme gene sequence of MS2 bacteriophage into the multiple cloning site 1 of pACYCDuet-1 vector plasmid by adopting a restriction enzyme cutting mode to obtain pACYC-MS2 vector plasmid; the conventional detection region sequence and the optimized packaging sequence of related viruses are inserted into a multiple cloning site 2 of a pACYC-MS2 vector plasmid in a restriction enzyme cutting mode, so that the vector can express MS2 capsid protein and exogenous viral RNA simultaneously, and the specific steps are as follows:

1) synthesis of the MS2 Gene sequence

The MS2 gene sequence was obtained from GenBank database, and BamHI and Not I restriction site sequences were designed and added to both ends of the sequence, and the designed sequence was artificially inserted into PUC-SP vector.

The MS2 gene sequence has a nucleotide sequence shown in SEQ ID NO. 1.

The MS2 gene sequences include gene sequences encoding the capsid protein and the maturase protein of the MS2 bacteriophage.

2) Construction and verification of pACYC-MS2 recombinant vector structure

Carrying out double digestion on the pACYCDuet-1 vector plasmid and the synthesized PUC-SP vector plasmid containing the MS2 gene sequence, adding BamHI and Not I restriction endonucleases, carrying out enzyme digestion for 1h at 37 ℃, and respectively carrying out gel recovery on target fragments after 1% agarose gel electrophoresis; subsequently, ligation was performed by T4 DNA ligase, the ligation product was transformed into TOP10 e. coli competent cells and cultured overnight, individual bacteria were picked for plasmid extraction, recombinant vector plasmid PCR validation, and further Sanger sequencing validation MS2 gene sequence was completely inserted into the multiple cloning site 1 of pacycuet-1 vector plasmid.

3) Synthetic virus recombinant conventional detection region sequence

Respectively screening out conventional detection regions of the seven RNA viruses, obtaining related sequence information from a GenBank database, designing and adding optimized MS2 phage packaging signals and enzyme cutting sites at two ends of the obtained sequence to form a recombinant detection sequence, and artificially synthesizing and inserting the designed sequence into a PUC-57 vector.

Preferably, the sequences of the conventional detection regions of the seven RNA viruses cover the target regions of most existing detection methods and detection kits.

Preferably, the conventional detection region sequences of the seven RNA viruses are linked at both ends to optimized MS2 phage packaging signals and restriction sites, and further form recombinant detection sequences.

Preferably, the optimized MS2 packaging signal has the nucleotide sequence shown in SEQ ID NO. 2.

Preferably, the recombinant detection sequence 1 of the respiratory syncytial virus type A (RSV-A) consists of an N gene, a P gene and an M gene fragment which are sequentially connected, the recombinant detection sequence 1 has a nucleotide sequence shown as SEQ ID NO.3, and enzyme cutting sites added at two ends are Kpn I and Pac I respectively; the recombination detection sequence 2 consists of a G gene, an F gene and an RdRp gene fragment which are sequentially connected, the recombination detection sequence 2 has a nucleotide sequence shown as SEQ ID NO.4, and enzyme cutting sites added at two ends are Kpn I and Fse I respectively.

Preferably, the recombinant detection sequence 1 of the respiratory syncytial virus B type (RSV-B) consists of an N gene, a P gene and an M gene fragment which are sequentially connected, the recombinant detection sequence 1 has a nucleotide sequence shown as SEQ ID NO.5, and enzyme cutting sites added at two ends are Kpn I and Pac I respectively; the recombination detection sequence 2 consists of a G gene, an F gene and an RdRp gene fragment which are sequentially connected, the recombination detection sequence 2 has a nucleotide sequence shown as SEQ ID NO.6, and enzyme cutting sites added at two ends are Kpn I and Pac I respectively.

Preferably, the recombinant detection sequence of rhinovirus C type (HRV-C) consists of 5' UTR gene, VP4 gene, VP2 gene and VP3 gene fragments which are sequentially connected, the recombinant detection sequence has a nucleotide sequence shown in SEQ ID NO.7, and enzyme cutting sites added at two ends are Kpn I and Pac I respectively.

Preferably, the recombination detection sequence 1 of parainfluenza virus type 1 (PIV-1) consists of HN gene segment, the recombination detection sequence has a nucleotide sequence shown as SEQ ID NO.8, and enzyme cutting sites added at two ends are Kpn I and Pac I respectively; the recombinant detection sequence 2 of the parainfluenza virus 1 consists of an N gene and an L gene segment, the recombinant detection sequence has a nucleotide sequence shown as SEQ ID NO.9, and enzyme cutting sites added at two ends are Kpn I and Pac I respectively.

Preferably, the recombination detection sequence 1 of parainfluenza virus type 3 (PIV-3) consists of HN gene segment, the recombination detection sequence has a nucleotide sequence shown as SEQ ID NO.10, and enzyme cutting sites added at two ends are Fse I and Pac I respectively; the recombinant detection sequence 2 of parainfluenza virus type 1 consists of N gene, M gene and L gene segment, the recombinant detection sequence has a nucleotide sequence shown as SEQ ID NO.11, and enzyme cutting sites added at two ends are Kpn I and Pac I respectively.

Preferably, the recombination detection sequence of the coxsackievirus A16 type (CA16) consists of a 5' UTR gene, a VP3 gene, a VP1 gene and a P2-A gene fragment which are connected in sequence, the recombination detection sequence has a nucleotide sequence shown as SEQ ID NO.12, and enzyme cutting sites added at two ends are Fse I and Pac I respectively.

Preferably, the recombination detection sequence of the enterovirus71 (EV71) consists of a 5' UTR gene, a VP3 gene, a VP1 gene and a P2-A gene fragment which are sequentially connected, the recombination detection sequence has a nucleotide sequence shown as SEQ ID NO.13, and enzyme cutting sites added at two ends are Fse I and Pac I respectively.

2) Construction and verification of recombinant vector plasmid

Carrying out double enzyme digestion on the pACYC-MS2 vector plasmid and the synthesized vector plasmids respectively containing the conventional detection regions of the seven RNA viruses, adding corresponding restriction endonucleases according to enzyme digestion sites added at two ends during synthesis, carrying out enzyme digestion at 37 ℃ for 1h, and carrying out gel recovery on target fragments respectively after 1% agarose gel electrophoresis; ligation was then performed by T4 DNA ligase, the ligation products were transformed into TOP10 e. coli competent cells and cultured overnight, individual bacteria were picked for plasmid extraction, recombinant vector plasmid PCR validation, and further Sanger sequencing validation.

The step 2 of plasmid transformation, induced expression and purification of the MS2 virus-like particles comprises the following steps:

1) successfully verified recombinant plasmids were transformed into BL21(DE3) E.coli competent cells, and recombinant plasmid positive bacteria were inoculated into LB liquid medium containing chloramphenicol resistance (final concentration 34. mu.g/mL);

2) culturing (37 ℃, 250rpm) until the OD value of the bacterial liquid is 0.6-0.8, adding an inducer IPTG (1mol/L) into the bacterial liquid to the final concentration of 1mmol/L, and inducing the expression of the recombinant MS2 virus-like particles;

3) the shaking culture was continued for about 12-16 hours, and the cells were collected by low-temperature centrifugation (4 ℃ C., 9500rpm), washed twice with 1 XPBS buffer, and added with sonication buffer (100mM NaCl, 5mM MgSO 5)₄50mM Tris base) and sonicated using a sonicator to release MS2 virus like particles (settings: the ultrasonic power is 15%, the crushing is carried out for 1.5s each time, the suspension is carried out for 3s), ice bath ultrasonic is carried out, ice is changed once per hour, and protein denaturation caused by high temperature generated during ultrasonic is avoided. After completion of the disruption, the resulting mixture was centrifuged at a high speed at a low temperature (4 ℃ C., 11,600rpm), and the supernatant was collected.

4) Solid sodium chloride (58.44g/mol) was added to the supernatant collected in the previous step to a final concentration of 1mol/L to dissolve the sodium chloride sufficiently, and then PEG6000 was added thereto to a final concentration of 10% (w/v), followed by shaking vigorously at 37 ℃ for 2min and standing in an ice bath for 6-8 hours or overnight. And (3) after low-temperature high-speed centrifugation, removing a supernatant, adding 1 XPBS for suspension precipitation, then adding isometric chloroform, violently shaking for 2min, carrying out low-temperature high-speed centrifugation for 15min, absorbing a lower-layer water phase, and concentrating by adopting PEG20000 to obtain the enriched MS2 virus-like particles.

5) Separating and purifying the PEG 6000-enriched MS2 virus-like particles by adopting propylene dextran Gel filtration chromatography (Bio-Gel A-1.5m Gel, Fine, Bio-Red) to remove impurity proteins, collecting a component flowing out of a first peak corresponding to a protein elution peak, namely the purified MS2 virus-like particles, and storing at 4 DEG C

Preferably, the enriched MS2 virus-like particles are loaded at a flow rate of 0.5mL/min, with the first peak appearing at about 60 min.

The step 3MS2 virus-like particle verification and quantification steps are as follows:

1) the purified MS2 virus-like particles were digested with DNase I and RNase A at final concentrations of 200U/mL and 500U/mL, respectively, digested in a 37 ℃ water bath for 2h to remove residual nucleic acids, and analyzed by 1% agarose gel electrophoresis.

The purified and digested MS2 virus-like particle is an RNA-protein complex of related virus recombinant RNA sequences wrapped by the capsid protein of the MS2 bacteriophage, and the MS2 virus-like particle digested by DNase I and RNase A can be seen as a bright single band.

2) MS2 phage coat protein analysis was verified using polyacrylamide gel electrophoresis (SDS-PAGE), consistent with conventional methods.

The purified and digested MS2 virus-like particle is an RNA-protein complex of a related virus recombinant RNA sequence wrapped by the capsid protein of the MS2 bacteriophage, and a single capsid protein monomer band can be observed at about 14kDa after heating lysis and SDS-PAGE electrophoretic staining.

3) RNA extraction and reverse transcription PCR amplification are carried out on the purified MS2 virus-like particles, and Sanger sequencing is carried out on the amplification products for verification; the MS2 virus-like particles successfully verified by Sanger sequencing were then subjected to real-time fluorescent reverse transcription PCR identification and droplet digital PCR absolute quantification.

The invention designs a group of primers and probes for respectively detecting the specificity of the seven RNA viruses, and the primers and probes can be used for detecting the real-time fluorescent reverse transcription PCR (polymerase chain reaction) method by a probe method and the droplet digital PCR method by the probe method, wherein the specific primers and probes are RSV-A (SEQ ID NO.3) and SEQ ID NO. 14-16; RSV-A (SEQ ID NO.4), SEQ ID NO. 17-19; RSV-B (SEQ ID NO.5), SEQ ID NO. 20-22; RSV-B (SEQ ID NO.6), SEQ ID NO. 23-25; HRV-C (SEQ ID NO.7), SEQ ID NO. 26-28; PIV-1(SEQ ID NO.8), SEQ ID NO. 29-31; PIV-1(SEQ ID NO.9), SEQ ID NO. 32-34; PIV-3(SEQ ID NO.10), SEQ ID NO. 35-37; PIV-3(SEQ ID NO.11), SEQ ID NO. 38-40; CA16(SEQ ID NO.12), SEQ ID NO. 41-43; EV71(SEQ ID NO.13), SEQ ID NO. 44-46. The detection primer and probe sequences are shown in Table 1 and the sequence table (SEQ ID NO.14-NO. 46).

TABLE 1 primer and Probe sequences

The step 4 of preparing the MS2 virus-like particle nucleic acid detection quality control product of the seven RNA viruses comprises the following steps:

1) diluting and subpackaging the sample after the identification and quantification are finished by adopting DMEM diluent, subpackaging the sample into 1mL of each tube, and storing at-80 ℃.

2) Sample freeze-drying (0.37mbar, 25 hours) is carried out by using Christ ALPHA 1-2 type freeze-drying machine, and the freeze-dried sample is stored at-20 deg.C, so as to avoid sample degradation caused by improper transportation and storage.

The invention adopts the method to prepare the MS2 virus-like particle nucleic acid detection quality control product of seven RNA viruses, and adopts DNase I and RNase A double-enzyme digestion, polyacrylamide gel electrophoresis, reverse transcription PCR, Sanger sequencing and real-time fluorescence reverse transcription PCR to verify the successfully prepared quality control product; the liquid drop digital PCR is adopted for absolute quantification of successfully prepared quality control products.

The invention relates to an application of the seven RNA virus nucleic acid detection quality control products in preparing laboratory quality control reagents.

A quality control reagent for RNA virus nucleic acid detection comprises seven RNA virus nucleic acid detection quality control products, which have nucleotide sequences shown in sequence tables SEQ ID NO.3-SEQ ID NO.13 and are MS2 virus-like particle nucleic acid detection quality control products of seven RNA viruses.

The quality control reagent further comprises primer probe sequences which have nucleotide sequences shown in sequence tables SEQ ID NO.14-SEQ ID NO. 46.

The invention has the beneficial effects that:

1) the MS2 virus-like particle nucleic acid quality control product of the seven RNA viruses prepared by the invention has high similarity with natural virus structures, stable performance, nuclease resistance and high safety, avoids the defect of high biological infection risk of clinical virus materials, and is basically equal to the virus materials; the quality control product can realize the quality monitoring and evaluation of the whole process of the extraction, reverse transcription and amplification detection of the seven viruses from the nucleic acid, and ensure the reliability of the detection result.

2) The quality control product has good universality and wide detection area, covers the target areas of most of the existing detection methods and detection kits, is suitable for the quality control of the conventional detection method and various detection kits, and overcomes the problem that the quality control product cannot be shared by different kits at present.

3) The MS2 virus-like particles prepared by the invention have wide application, can be used for routine indoor quality control, and can also be used for laboratory detection process optimization, kit performance confirmation, indoor evaluation and as a positive control of a virus detection kit. And can also be used for quality control of virus multiplex joint inspection by mixing different virus MS2 virus-like particles.

4) The preparation method is simple and efficient. Adopts a single particle double expression system to overcome doubleThe plasmid expression system has low preparation efficiency, and the sequence optimization can realize the high-efficiency packaging of sequences larger than 1800 bp; through subsequent purification and digestion steps, the high-purity and high-concentration MS2 virus-like particles can be prepared and obtained, and the concentration can reach 10¹²copies/mL, single preparation volume can meet the detection of thousands of laboratories. In addition, the MS2 virus-like particles are prepared into a freeze-dried product by using a vacuum freeze-drying technology, so that the performance of the freeze-dried product is more stable, and the freeze-dried product is easier to store and transport for a long time.

5) The invention also provides a primer and a probe sequence for detecting the specificity of the seven RNA viruses, and the primer and the probe sequence can be used for detecting the real-time fluorescent reverse transcription PCR by a probe method and the droplet digital PCR by the probe method.

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below by taking the EV71 virus as an example, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and those skilled in the art can also obtain other drawings based on these drawings without inventive labor.

Drawings

FIG. 1 is a schematic diagram of the structure of a double expression vector constructed according to the present invention.

FIG. 2 shows the results of purification of EV71 MS2 virus-like particles by Sephadex propylene chromatography.

FIG. 3 is the result of nuclease digestion of EV71 MS2 virus-like particles; wherein, M is 2000bp DNA Marker, and Lane 1 is EV71 MS2 virus-like particles after DNase I and RNase A digestion; lane 2 is EV71 MS2 virus-like particles not digested with DNase I and RNase A.

FIG. 4 shows the result of polyacrylamide gel electrophoresis of EV71 MS2 virus-like particles, wherein M is 170kDa protein Marker, and Lane 1 is the cleaved EV71 MS2 virus-like particles.

FIG. 5 shows the result of RT-PCR electrophoresis of EV71 MS2 virus-like particles, in which M is 2000bp DNA Marker, and lane 1-2 is one-step RT-PCR amplification product after RNA extraction from EV71 MS2 virus-like particles.

FIG. 6 is EV71 MS2, performing gradient dilution on the virus-like particles to obtain a real-time fluorescence reverse transcription PCR result; wherein, the virus-like particle is EV71 MS2 virus-like particle (10) after nuclease digestion from left to right³To 10⁷Fold gradient dilution) results of RNA extraction amplification after reverse transcription, direct amplification of unextracted RNA of EV71 MS2 virus-like particles after nuclease digestion, and negative control (no template) amplification.

FIG. 7 shows the results of droplet digital PCR quantification of EV71 MS2 virus-like particles.

FIG. 8 shows the validation results of the commercial kit for EV71 MS2 virus-like particles

Detailed Description

In order that the invention may be more readily understood, the following detailed description of the invention is given in conjunction with specific examples, it being understood that the examples described are only a part of the invention, and not all of them, and are not intended to limit the scope of the invention in any way. Any equivalent substitutions made in accordance with the present disclosure are intended to be within the scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The main materials and reagents used in the following examples are as follows:

plasmid pACYCDuet-1 was purchased from Novagen.

The product strains Top10, BL21(DE3) were purchased from Tiangen.

The plasmid miniprep kit and the DNA recovery kit are purchased from Tiangen company.

T4 DNA ligase, restriction enzyme was purchased from Thermo Fisher Scientific.

QIAamp Viral RNA Mini Kit, QIAGEN OneStep RT-PCR Kit was purchased from QIAGEN.

Digital PCR reagents were purchased from Bio-Rad.

EXAMPLE 1 construction of pACYC-MS2-virus vector construction

1. Construction of pACYC-MS2 recombinant plasmid

1) Obtaining gene sequences for coding the capsid protein and the mature enzyme protein of the MS2 bacteriophage from a GenBank database, designing and adding BamH I and Not I restriction enzyme cutting site sequences at two ends of the sequences, and displaying sequence information as SEQ ID No. 1; the designed sequence was artificially synthesized by Biotechnology engineering (Shanghai) Co., Ltd and inserted into the PUC-SP vector.

2) The PUC-57 and pACYCDuet-1 plasmids were double digested with restriction enzymes BamHI and Not I, respectively, as follows:

TABLE 2

Enzyme digestion is carried out for 1h at 37 ℃, restriction enzyme is inactivated at 80 ℃ for 5min, the enzyme digestion target products are respectively subjected to 1% agarose gel electrophoresis, gel cutting, recovery and purification are carried out, and the products are respectively named as MS2-s and pACYC-l.

4) Connecting the recovered enzyme digestion products MS2-s and pACYC-l by using a T4 DNA ligase kit, wherein the connecting system is as follows:

TABLE 3

5) Incubating and connecting at 22 ℃ for 30min, transforming TOP 10E. coli competent cells by a connecting product, plating, selecting a monoclonal colony for PCR identification, correctly identifying by PCR, extracting plasmid sequencing, and naming pACYC-MS2 by correctly sequencing the plasmid, thereby inserting the MS2 gene sequence into the polyclonal site 1 of the pACYCDuet-1 vector.

2. Construction of pACYC-MS2-virus recombinant plasmid

1) Screening out conventional detection regions of the seven RNA viruses respectively, obtaining related sequence information from a GenBank database, designing and adding optimized MS2 phage packaging signals and enzyme cutting sites at two ends of the obtained sequence to form a recombinant detection sequence, wherein the sequence information is shown as SEQ ID No.3-No. 13; the designed sequence was artificially synthesized by Biotechnology engineering (Shanghai) Co., Ltd and inserted into the PUC-57 vector, which was named PUC-57-virus (corresponding virus name).

2) The PUC-57-virus (corresponding virus name) and pACYC-MS2 plasmids were double-digested with the corresponding restriction enzymes as follows:

TABLE 4

Enzyme digestion is carried out for 1h at 37 ℃, restriction enzyme is inactivated at 80 ℃ for 5min, the enzyme digestion target products are respectively subjected to 1% agarose gel electrophoresis, gel cutting, recovery and purification, and the products are respectively named as virus-s (corresponding virus name) and pACYC-MS 2-l.

4) And (3) connecting the recovered enzyme digestion products, namely viruses-s (corresponding to the virus names) and pACYC-MS2-l by using a T4 DNA ligase kit, wherein the connecting system is as follows:

TABLE 5

5) Incubating and connecting at 22 ℃ for 30min, transforming TOP 10E. coli competent cells by a connecting product, plating, selecting a monoclonal colony for PCR identification, carrying out PCR identification on a correct extracted plasmid for sequencing, and naming pACYC-MS 2-viruses (corresponding virus names) as a correct sequencing plasmid, so that a recombinant conventional detection region sequence of the virus is inserted into a multiple cloning site 2 of a pACYCDuet-1 vector, and a structural pattern diagram of a finally constructed double expression vector is shown as an attached figure 1 of the specification.

EXAMPLE 2 preparation of MS2 Virus-like particles containing seven RNA viral nucleic acids

1) pACYC-MS2-virus recombinant plasmids of seven viruses successfully sequenced were transformed into expression-type BL21(DE3) E.Coli competent cells, respectively.

2) The positive clone is inoculated into 5mL LB liquid culture medium containing chloramphenicol (with the final concentration of 34 mug/mL), cultured for about 4-6h, 2mL of bacterial liquid is added into 200mL of LB liquid culture medium for amplification culture, the culture is carried out at 37 ℃ and 250rpm for about 2-3h by oscillation, when the OD 600 value of the bacterial liquid is detected by a spectrophotometer to be about 0.6-0.8, isopropylthio-beta-D-galactoside (IPTG) with the final concentration of 1mmol/L is added into the bacterial liquid, the culture is carried out at 37 ℃ and 250rpm for about 12-16 h by oscillation, and the expression of MS2 virus-like particles is induced.

3) Centrifuging at 9500rpm for 3min at 4 deg.C, discarding the supernatant, retaining the bacterial pellet, suspending and washing the bacterial pellet twice with 1 XPBS, discarding the supernatant, and adding sonication buffer (100mM NaCl, 5mM MgSO 5)₄50mM Tris base) 30mL of resuspended pellet, sonicated using a sonicator to release MS2 virus-like particles (settings: the ultrasonic power is 15%, the crushing is carried out for 1.5s each time, the suspension is carried out for 3s), ice bath ultrasonic is carried out, ice is changed once per hour, and protein denaturation caused by high temperature generated during ultrasonic is avoided.

4) After the bacteria crushing is finished, centrifuging at the temperature of 4 ℃ and the rpm of 11,600 for 20-30min, and collecting supernatant.

5) Solid sodium chloride (58.44g/mol) was added to the supernatant collected in the previous step to a final concentration of 1mol/L to dissolve the sodium chloride sufficiently, and then PEG6000 was added thereto to a final concentration of 10% (w/v), followed by shaking vigorously at 37 ℃ for 2min and standing in an ice bath for 6-8 hours or overnight.

6) Centrifuging at 4 ℃ and 11,600rpm for 15min, discarding the supernatant, adding 5mL of 1 XPBS for suspension precipitation, then adding equal volume of chloroform, violently shaking for 2min, centrifuging at 4 ℃ and 11,600rpm for 15min, sucking the lower aqueous phase, and concentrating by using PEG20000 to obtain the enriched MS2 virus-like particles.

7) Separating and purifying the MS2 virus-like particles enriched with PEG6000 by adopting propylene dextran Gel filtration chromatography (Bio-Gel A-1.5m Gel, Fine, Bio-Red) to remove impurity proteins, sucking 5mL of MS2 virus-like particles to be purified by using a clean disposable 5mL syringe, filtering and sterilizing the particles by using a 0.22 mu m filter, loading the particles with the loading amount of 5mL, purifying the sample at the flow rate of 0.5mL/min, and collecting components flowing out of a first elution peak (shown in the specification and attached figure 2) by using a collection tube for about 60min, wherein each tube is 1 mL. Namely the purified MS2 virus-like particles, and the purified MS2 virus-like particles are preserved at 4 ℃.

Example 3MS2 verification and quantification of Virus-like particles

1. Enzyme digestion verification of DNase I and RNase A

The MS2 virus-like particle solution obtained in example 2 was digested with DNase I and RNase A at final concentrations of 200U/mL and 500U/mL, respectively, and the sample addition system was as follows:

TABLE 6

The residual nucleic acids were removed by digestion in a 37 ℃ water bath for 2h and analyzed by 1% agarose gel electrophoresis.

By taking EV71 virus as an example, and performing electrophoretic analysis (as shown in the attached figure 3 of the specification), it can be seen that the EV71 MS2 virus-like particle purified in example 2 is a bright single band after being digested by DNase I and RNase A, while the virus-like particle without being digested by DNase I and RNase A has a "tailing phenomenon" due to the adhesion of nucleic acid remained on the surface, which indicates that the EV71 MS2 virus-like particle purified in example 2 is a protein-nucleic acid complex with a specific size and is resistant to DNase and RNase attacks at the same time, and the same is consistent with the expected result for other virus MS2 virus-like particles.

2. Polyacrylamide gel electrophoresis (SDS-PAGE) validation

MS2 phage capsid protein analysis by SDS-PAGE confirmed the use of 5% concentrated gel and 15% split gel, consistent with conventional methods.

The capsid protein of the MS2 bacteriophage consists of 180 monomeric proteins of about 14kDa in size, the outer capsid protein of which is cleaved into monomeric proteins when denatured by heat. For example, by using EV71 virus as an example, a monomer band can be observed at about 14kDa by electrophoretic staining analysis (as shown in the attached figure 4 of the specification), which indicates that the externally-wrapped protein is the MS2 capsid protein, and the other viruses are MS2 virus-like particles.

3. Reverse transcription PCR and Sanger sequencing verification

The MS2 virus-like particles digested in step 1 of example 3 were subjected to RNA extraction using the QIAamp Viral RNA Mini Kit, and to one-step RNA reverse transcription PCR using the QIAGEN OneStep RT-PCR Kit, using the following loading system:

TABLE 7

The reaction conditions are as follows: reverse transcription is carried out for 30min at 50 ℃; pre-denaturation at 95 ℃ for 15 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 2min, and 30 cycles; final extension at 72 ℃ for 10 min.

The amplification products were analyzed by 1% agarose gel electrophoresis and confirmed by Sanger sequencing.

By taking EV71 virus electrophoretic analysis (as shown in the attached figure 5 of the specification) as an example, the EV71 virus recombination detection sequence can be amplified by reverse transcription PCR, and the amplification band and the sequencing result are consistent with expectations, which shows that the EV71 virus recombination detection sequence is successfully wrapped in the MS2 capsid protein, and the other viruses are MS2 virus-like particles.

4. Real-time fluorescent reverse transcription PCR validation

RNA was extracted by 10-fold gradient dilution of nuclease-digested MS2 virus-like particles obtained in step 1 of example 3, and the RNA was purified according to PrimeScript^TMThe cDNA was obtained by reverse transcription using the RT Master Mix (Perfect read Time) instructions, and the loading system was as follows:

TABLE 8

The reaction conditions are as follows: reverse transcription at 37 deg.C for 15 min; reverse transcriptase was inactivated at 85 ℃ for 5 sec.

The reverse transcription cDNA and the digested MS2 virus-like particles are detected by a Takara Premix Ex TaqTM (Probe rRT-PCR) fluorescent Probe PCR detection reagent, the sequences of the adopted primers and probes are shown in the specification table 1, and the sample adding system is as follows:

TABLE 9

The reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 sec; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s (fluorescence collection), for 40 cycles.

Taking an EV71 virus fluorescence detection result (shown in figure 6 in the specification accompanying drawing) as an example, RNA extracted after 10-fold gradient dilution of EV71 MS2 virus-like particles digested by nuclease can be detected, the difference of gradient samples is about 3.3 Ct values, and the difference is consistent with an expected result, which indicates that the EV71 virus recombination detection sequence is wrapped by the MS2 virus-like particles; and under the same concentration level (diluted by 1000 times), the Ct value directly detected by the MS2 virus-like particle protein after nuclease digestion is 38, while the Ct value after RNA extraction and fluorescence reverse transcription PCR of the MS2 virus-like particle after nuclease digestion is 19, which indicates that the residual nucleic acid in the MS2 virus-like particle solution is completely digested.

5. Droplet digital PCR quantification

After RNA extraction and reverse transcription are carried out on the MS2 virus-like particles digested by nuclease in the step 1 of example 3, absolute quantification of MS2 virus-like particles is carried out by adopting a probe method and droplet digital PCR, the sequence of the adopted primer and probe is shown in the specification table 1, a probe method premix is selected, a 20uL probe method quantitative reaction system is prepared for microdroplet generation, and the reaction system is shown as follows:

watch 10

After the microdroplets were generated, the microdroplets were transferred to a 96-well plate and sealed for PCR reaction.

The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30s, annealing/extension at 60 ℃ for 1min for 40 cycles; inactivating thermal kinase at 98 deg.C for 10min, and storing at 4 deg.C.

For example, using EV71 virus as an example, an EV71 original-fold MS2 virus-like particle sample is diluted by 1 × 10⁵After doubling, the solution is diluted again in a doubling ratio, and the result of the digital PCR detection of the liquid drops is obtained (as shown in the attached figure 7 of the specification)Shown) it can be seen that the quantitative result is a fold-rate change, and the quantitative result of the diluted sample is 9.5X 10⁷copies/mL, therefore EV71 original times MS2 virus-like particle is 9.5X 10¹²copies/mL, it can be seen that the concentration of MS2 virus-like particles prepared by the method of the present invention is much higher than that of other methods.

Example 4MS2 practical detection of Virus-like particles

The seven RNA viruses MS2 virus-like particles quantified in example 3 were practically detected using a commercial kit. Using EV71 as an example, MS2 virus-like particles quantified in example 3 were diluted to 1X 10⁷The copies/mL is detected by adopting an enterovirus EV71/CA16/EV nucleic acid detection kit (PCR-fluorescent probe method) of Daan GenBank, Zhongshan university, and a detection method and judgment rule reference kit, if a detection sample has an amplification curve in an FAM channel and a Ct value is less than or equal to 44, a Texas Red channel has an amplification curve and a Ct value is less than or equal to 44, and a CY5 detection channel has or does not have an amplification curve, the sample can be judged to be EV71 positive.

The detection result of the kit (as shown in figure 8 in the specification) shows that the EV71 MS2 virus-like particles have an amplification curve in an FAM channel, the Ct value is 19, the Texas Red detection channel has an amplification curve, the Ct value is 29, and the EV71 virus-like particles can be judged to be positive according to the judgment standard, which shows that the prepared EV71 MS2 virus-like particles are suitable for laboratory detection quality control and used as a positive control of a conventional kit, and other virus MS2 virus-like particles also can be used.

Example 5 nucleic acid detection and quality control reagents for seven RNA viruses

The virus-like particles MS2 (SEQ ID NO.3-SEQ ID NO.13) of the seven RNA viruses quantified in example 3 were diluted with DMEM diluent to 1X 10⁵Subpackaging copies/mL into 1 mL/tube, lyophilizing with Christ ALPHA 1-2 type lyophilizer, and storing the lyophilized quality control product at-20 deg.C; the virus-specific primer probe (SEQ ID NO.14-SEQ ID NO.46) was diluted with ribozyme-free water to 10. mu.M, split-packaged into 100. mu.L/tube, and stored at-20 ℃ in the dark. Packaging the prepared quality control product and the primer probe into a set to obtain the nucleic acid detection quality control reagent for the seven RNA viruses.

The invention has strong industrial practicability, and can obtain the nucleic acid detection quality control reagent of seven RNA viruses through simple operations such as dilution, split charging, freeze drying and the like.

The series of detailed descriptions set forth herein are merely specific to possible embodiments of the invention and are not intended to limit the scope of the invention, as those skilled in the art will be able to devise numerous other modifications and embodiments that will fall within the scope and spirit of the principles disclosed herein. More specifically, various variations and modifications are possible in the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure, the drawings and the appended claims. In addition to variations and modifications in the component parts and/or arrangements, other uses will also be apparent to those skilled in the art.

Sequence listing

<110> Beijing Hospital

<120> seven RNA virus nucleic acid detection quality control product and preparation method thereof

<130> YRSW21I108

<141> 2021-08-05

<160> 46

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1727

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gggtgggacc cctttcgggg tcctgctcaa cttcctgtcg agctaatgcc atttttaatg 60

tctttagcga gacgctacca tggctatcgc tgtaggtagc cggaattcca ttcctaggag 120

gtttgacctg tgcgagcttt tagtaccctt gatagggaga acgagacctt cgtcccctcc 180

gttcgcgttt acgcggacgg tgagactgaa gataactcat tctctttaaa atatcgttcg 240

aactggactc ccggtcgttt taactcgact ggggccaaaa cgaaacagtg gcactacccc 300

tctccgtatt cacggggggc gttaagtgtc acatcgatag atcaaggtgc ctacaagcga 360

agtgggtcat cgtggggtcg cccgtacgag gagaaagccg gtttcggctt ctccctcgac 420

gcacgctcct gctacagcct cttccctgta agccaaaact tgacttacat cgaagtgccg 480

cagaacgttg cgaaccgggc gtcgaccgaa gtcctgcaaa aggtcaccca gggtaatttt 540

aaccttggtg ttgctttagc agaggccagg tcgacagcct cacaactcgc gacgcaaacc 600

attgcgctcg tgaaggcgta cactgccgct cgtcgcggta attggcgcca ggcgctccgc 660

taccttgccc taaacgaaga tcgaaagttt cgatcaaaac acgtggccgg caggtggttg 720

gagttgcagt tcggttggtt accactaatg agtgatatcc agggtgcata tgagatgctt 780

acgaaggttc accttcaaga gtttcttcct atgagagccg tacgtcaggt cggtactaac 840

atcaagttag atggccgtct gtcgtatcca gctgcaaact tccagacaac gtgcaacata 900

tcgcgacgta tcgtgatatg gttttacata aacgatgcac gtttggcatg gttgtcgtct 960

ctaggtatct tgaacccact aggtatagtg tgggaaaagg tgcctttctc attcgttgtc 1020

gactggctcc tacctgtagg taacatgctc gagggcctta cggcccccgt gggatgctcc 1080

tacatgtcag gaacagttac tgacgtaata acgggtgagt ccatcataag cgttgacgct 1140

ccctacgggt ggactgtgga gagacagggc actgctaagg cccaaatctc agccatgcat 1200

cgaggggtac aatccgtatg gccaacaact ggcgcgtacg taaagtctcc tttctcgatg 1260

gtccatacct tagatgcgtt agcattaatc aggcaacggc tctctagata gagccctcaa 1320

ccggagtttg aagcatggct tctaacttta ctcagttcgt tctcgtcgac aatggcggaa 1380

ctggcgacgt gactgtcgcc ccaagcaact tcgctaacgg ggtcgctgaa tggatcagct 1440

ctaactcgcg ttcacaggct tacaaagtaa cctgtagcgt tcgtcagagc tctgcgcaga 1500

atcgcaaata caccatcaaa gtcgaggtgc ctaaagtggc aacccagact gttggtggtg 1560

tagagcttcc tgtagccgca tggcgttcgt acttaaatat ggaactaacc attccaattt 1620

tcgctacgaa ttccgactgc gagcttattg ttaaggcaat gcaaggtctc ctaaaagatg 1680

gaaacccgat tccctcagca atcgcagcaa actccggcat ctactaa 1727

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

acatgaggat cacccatgt 19

<210> 3

<211> 2498

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cggggtacca catgaggatc acccatgtcc ttgatttcac agggtgtggt tacatcatat 60

gctagtttgc ttctttcatc caaggacaca ttagcgcata tggtaaattt gctgggcatt 120

tgtgctagca ctgcacttct tgagtttatc atgactctta gtgaaggtcc cttgggtgtg 180

gatatttgtt tcactagtat gttgacatta gctagttctt ttataagtaa atctgctggc 240

atagatgatt ggaacatggg cacccatatt gtaagtgatg cagggtcatc gtctttttct 300

aagacattgt attgaacagc agctgtgtat gtggagcctt cgtgaagctt gttcacgtat 360

gtttccatat ttgccccacc ctttcctttt tttgtaacta tattatagat tttttccggg 420

tggttagttt tggattggct ggttgttttg ttggctgttt ggctgattgg cggatggatg 480

tttggttgga tgattgggtt ggttagtttg ttggtcttct gttggtattg tgtgttgatg 540

tgaagagtgg taactaatca gaaatcttaa tgtgtgaagc attcctagta tttcacttaa 600

tttttcatca atcctatcta atcttgctgt tatattatcg tttgtctgat catttatttc 660

ttcgtatgaa tagctggatt cttcttcatt gttatcaaat gtttctatgg tttctttgta 720

tagtttagaa aagggattat cacttggtgt agggtcttct ttgaaactta ctagaggttt 780

tctttgataa ttgggcttgt tccctgcagt atcatctgtc tcatttgttg ggttgataat 840

agttgaattt gatgttatag ggctttcttt ggttacttct atatctattg agttgacaga 900

tatgatacta tcttttttct tgggatcttt gggtgatgtg aatttgccct ttattgattc 960

taggaattta gtagccctgt tgtttgcatc ttctccatgg aattcaggag caaacttttc 1020

catgatgatt tatttgcccc attttttatt aactcaaagc tctacatcat tatcttttgg 1080

attaagctga tgtttgatag cctctagttc ttctgctgtc aagtctagta cactgtagtt 1140

aatcacacca ttttctttga gttgttcagc atatgccttt gctgcatcat atagatcttg 1200

attcctcggt gtacctctgt actctcccat tatgcctagg ccagcagcat tgcctaatac 1260

tacactggag aagtgaggaa attgagtcaa agataataat gatgcttttg ggttgttcaa 1320

tatatggtag aatcctgctt caccacccaa tttttgggca tattcataaa cctcaacaac 1380

ttgttccatt tctgcttgca cactagcatg tcctaacata atatttttaa ctgattttgc 1440

taagactccc caccgtaaca tcacttgccc tgcaccatag gcattcataa acaatcctgc 1500

aaaaatccct tcaactctac tgccacctct ggtagaagat tgtgctatac caaaatgaac 1560

aaaaacatct ataaagtggg gatgtttttc aaacacttca tagaagctgt tggctatgtc 1620

cttgggtagt aagcctttgt aacgtttcat ttcatttttt aggacattat tagctctcct 1680

aatcacggct gtaagaccag atctgtcccc tgctgctaat ttagttatta ctaatgctgc 1740

tatacataat attatcatcc cacaatcagg agagtcatgc ctgtattctg gagctacctc 1800

tcccatttct tttagcattt ttttgtagga ttttctagat tctatctcaa tgttgatttg 1860

aatttcagtt gttaagcttg ccaatgttaa cacttcaaat ttcatttctt ttccattaat 1920

gtcttgacga tgtgttgtta catctactcc atttgctttt acatgatatc ccgcatctct 1980

gagtattttt atggtgtctt ctcttcctaa cctagacatc gcatataaca tacctattaa 2040

cccagtgaat ttatgattag catcttctgt gattaataac atgccacata acttattgat 2100

gtgtttctgc acatcataat taggagtatc aatactatct cctgtgctcc gttggatggt 2160

gtatttgctg gatgacagaa gttgatcttt gttgagtgta tcattcaact tgactttgct 2220

aagagccatc tttgtatttg ccccatcttc tatcttatat ctctccttaa ttttaaatta 2280

ctataatttt caggctccat ctggactatg gagtatagtt atgcatagag ttgttgtttt 2340

agattgtgtg aatattgtgt tgaaatttat ggattgagat catacttgta tattatggga 2400

gtatgctttg taggcttaat gccaatgcat tctaagaacc catcatgatt gatgaatatt 2460

ggcataggga catgaggatc acccatgttt aattaagg 2498

<210> 4

<211> 2318

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cggggtacca catgaggatc acccatgtta tgatcattgc aatctttcag acttctgtaa 60

atatatctta tgtcaggatg aagttccact actgtacgca ataataaatt ccctgctcct 120

tcacctatga atgctataca attgggatct ttaattttaa gatcttttaa aatatactct 180

atactaattt tacaacctgt agaactaaat acaaaattga atctattaat atgatgccaa 240

ggaagcatgc agtaaagtga tgtgctattg tgcactaatt gttctactac tgacattaaa 300

ctaaggccaa agcttataca gttttggaat actatgtcaa tatcttcatc accatacttt 360

tctgttaata tgcgattaat agggctagtg tcaaagtgat aatttgttgt tctataagct 420

ggtattgatg cagggaattc acatggtcta ctactgactg taaggcgatg caaataattg 480

acacttaaat attgtggaaa taatttcttg gccttttcat atgttaaccc aagggttcct 540

atgctgagtt cttccatgaa ttcatccttg ttatctattt ttatataact ataaactagg 600

aatctactta aatagtgtaa gtgagatggt ttatagatga gagtttcgat gaagttcaga 660

ttttaagaaa atccaatgac agatgggttg tctatgagca gatagtaaac cattgtaaga 720

acatgattag gtgctatttt tatttagtta ctaaatgcaa tattatttat accactcagt 780

tgatctttgc ttagtgtgac tggtgtgctt ctggccttac agtataagag cagtccaaca 840

gcaattaatg ataacaatat tactataatc actataatta tagtagttat catgatattt 900

gtggtggatt taccagcatt tacattatgt aataattcat cggatttacg aataaatgct 960

aggctctggt taatcttctc gttgacttga gatattgatg catcaaattc atcagagggg 1020

aatactaatg ggtcatagaa atttattatt ggttcacctt ttacatagag acttttacct 1080

tcttgcttat ttacataata taatgtgtta cctacagaca cagtgtccat ccctttattt 1140

gatacataat cgcacccgtt agaaaatgtc tttatgattc cacgattttt attggatgct 1200

gtacatttag ttttgccata gcatgacaca atggctccta gagatgtgat aacggagctg 1260

cttacatctg tttttgaagt cataatttta caatcatatt tggggttgaa tatgtcaaca 1320

ttgcagagat ttatttcact tggtaatgtt aaactgttca ttgtgtcaca aaatactcga 1380

tttgattgaa ctttacatgt ttcagcttgt gggaagaaag atactgatcc tgcattgtca 1440

cagtaccatc ctctgtcagt tcttgttaaa cagatgttgg acccttcttt tgtgttggtt 1500

gtacatagag gggatgtgtg tagtttccaa cagggtgtat ctataacacc atatagtggt 1560

aattgtacta catatgctaa gacttcctct tttattatgg acatgataga gtaactttgc 1620

tgtctaacta tttgaacatt gttggacatt aactttttct gatcatttgt tataggcata 1680

tcattgatta atgacaataa ttcactatta gttaacatgt aagtgcttac aggtgtagtt 1740

acacctgcat taacactaaa ttccctggta atctctagta gtctgttgtt cttttgttgg 1800

aactctatca cagtttctat atttgatata cggattagag ggactgattc caagctgagg 1860

attctgggtg aggtatgttg gggttgtgtt cttgatctgg cttgttgcat cttgtatgat 1920

tgcagttgtt ggtgtgactt tgtggtttgc cgaggctatg aatatgatgg ctgcaattat 1980

aagtgaagtt gagattatca ttgccagaat ggataatgtg atttgtgcta cagatttaag 2040

atttaactta tataagcacg atgatatgaa taataaatga ttgagagtgt cccaggtcct 2100

ttctaatgtc ttagcggtgc gttggtcctt gtttttggac atgtttgcat ttgccccaat 2160

gttattgtta gtcttgatat cctagttcat tgttatgact atttttaatt aactacttta 2220

tagtatggat agtggtttgc atggtgggat gttaatgagg tgttgtaaag aggtaggggt 2280

tgttcattta catgaggatc acccatgtgg ccggcccc 2318

<210> 5

<211> 2058

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cggggtacca catgaggatc acccatgtct tccgctctta ttttttctat catttcttct 60

ctcagaccaa ccatagcatc tcttattcca tcgcgagctg aagtgggtcc tgcacttgca 120

actactaatg tatggagcat tcctaatatt tcacttaatt tttcatcaat tctatctagt 180

cttgctgtaa tgttgtcatt tgtttgatca tttatctctt catatgagta gctagattct 240

tcttcattgt tatcaaatgt ttctattgtt tctttgtaca acttagaaaa agggttgtca 300

cttggggtga gatcttcttt gaagcttact aggggttttc ttgggtagtt ggctttggtt 360

tctggggtac tgtcggcttc acttgttgga ttgatgatgt tggtgccaga tgttatcggg 420

ctctctttgg ttacttctat atctattgag ttaacagata ttatgctatc tttcttctta 480

ggatctttgg atgatgcgaa cttgcccttt attgattcta ggaatttggt agctttgtta 540

tttgcatctt ctccatgaaa ttcaggtgca aacttctcca tgttgactta tttgccccgt 600

attttttgtt aacttaaagc tctacatcat cttctttagg gttgagttga ttctttatgg 660

cttccaattc ttctgctgtt aagtctaata cactgtagtt tattactcca ttttctttga 720

gttgctctgc atatgctttg gctgcatcat aaagatcctg gtttcttggc gtacctctat 780

actctcccat tatgcctaga cctgctgcat tgcctaggac cacacttgag aagttaggaa 840

attgagttaa tgacagcaat gatgcttttg gattgttcaa tatatggtag aatccagctt 900

ctcctcccaa cttctgtgca tactcataga cttccacaac ttgctccatt tctgcctgga 960

cactagcatg acctagcatg atatttttta cagatttggc taaaactccc catcttagca 1020

ttacttgccc tgaaccatag gcattcataa acaatcctgc aaagattcct tcaactctac 1080

taccccctct tgttgatgat tgtgcaatgc caaagtgcac aaaaacatct ataagatgag 1140

ggtgtttttc aaacacttca taaaaactgt tagctatatc ctttggtatg agacccttgt 1200

agcgttttat ttcatttttt aagacattgt ttgccctcct aattactgct gtaagacctg 1260

atctgtctcc tgctgctaat ttggttatta caagtgctgc tatacacagt attatcatcc 1320

cacagtctgg agaatcatgc ctatattctg gagccacttc tcccatctct tttagcattt 1380

ttttgtagga ttttctagat tctatctcaa tattgacttg tatttctgat gtcaagcttg 1440

ataatgttaa tacttcgaat ttcatttcct ttccatttat atcttgacga tatgttgtta 1500

tatctactcc attagcttta acatgatatc cagcatcttt aagtatcttt atagtgtctt 1560

cccttcctaa cctggacata gcatataaca tacctattaa tcctgtgaat ttatgatttg 1620

catcttcagt gattaatagc ataccacata gtttgtttag gtgtttttgc acatcataat 1680

tgggagtgtc aatattatct cctgtactac gttgaatagt gtatttgctg gatgacagca 1740

gctgatcctt atttaatgta tcatttaact tgactttgct aagagccatc tttgtatttg 1800

ccccaattta tgttattggc tttactttta tttttaatta ctaaaacgga ttagctcctt 1860

cttaactatt gagcattgtt gtttgaggaa tagcttggtt tggttgggtt ggtttttttt 1920

gttggaattt acgggttgag gtcatatttg tatattatag gagtgtgttt tgtaggctta 1980

atgccaatac attctagaaa cccgccatga ttgataaata taggcatgga catgaggatc 2040

acccatgttt aattaagg 2058

<210> 6

<211> 2409

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cggggtacca catgaggatc acccatgtta tgcccgttgt ataatcttag aaattcaata 60

ggtaaactat gatcattgca atcttttaaa cttctgtaaa tgtatcttat gtctggatga 120

agttctacta ccgtacgtaa taataagtta ccagctcctt cacctatgaa tgctatacaa 180

ctggggtcct taatcttaag atcttttaaa atatactcta tactgatctt gcatcctgtg 240

gaactaaata caaagttaaa tctattgaca tgatgccaag gaagcatgca ataaagtgat 300

gcactattcc ttactaaaga tgtctgatcc gacatcaggc taagaccaaa acttatgcaa 360

ttttgaaaca caatgtcgat atcttcatct ccatactttt ctgttaatac atgattgata 420

ggactagtat cgaaatgata atttgttgtt ctataagctg gtattgatgc agggaattca 480

catggtctac tactgactgt taaacggtgt aaataattga cacttagata ttgtggaaac 540

aactttttgg ctttttcata tgacagtcca agtgttccag tactcagttc ttccatgaat 600

tcatctttgt tgtctatgga tgcatatatg tgaagaaatg attcagcctg tactgtgatg 660

ttgaagtgtt tgttatgggt tgatttggga ttggtgatca gcagactgtt gttgaaacat 720

gatcaggtgg ttttttgtct atttgctgaa tgcaatatta ttgattccac ttagttggtc 780

tttgcttagt gtaactggtg tgtttttggc tttgcaatac aacagcaaac caatagctat 840

taatgataac aatactacaa tgattactat aataattgta gttatcataa tatttgtagt 900

agatttgcca gtatttacat tatgtagtaa ttcatcagat ctacgaataa aagctaaact 960

ttgattgatt ttttcattga cttgagatat tgatgcatca aactcatcag aaggaaacac 1020

tagagggtca tagtaattta ttataggttc cccttttaca taaaggttct tgccttccag 1080

cttgtttaca tagtataaag tgttgcccac tgacacagta tctactcctt tgtttgacac 1140

atagtcacaa ccattagaaa atgtctttat aatcccacga tttttgttgg atgcagtgca 1200

tttagtttta ccatagcatg acactatagc tccaagagaa gtaattactg agctgcttat 1260

gtctgttttt gatgtcataa ttttgcagtc atacttggaa ttgaatatgt cagtgttaca 1320

aaggctgact tcacttggta atgtcaaact gttcatagtg tcacaaaata ctcgattgga 1380

ctgtacttta caagtgtcag cctgtggaaa gaaggatact gatcctgcat tatcacaata 1440

ccatcctcta tcagtccttg ttaaacaaat atttgatcct tctttgatgt tggtggtgca 1500

tagaggtgat gtgtgtaatt tccagcaagg tgtatctatt acaccataga taggtagctg 1560

tacaacatat gcaaggactt cttcctttat tatagacatg atagaataac tttgttgcct 1620

tactatctga acattgcttg acattaattt tttctgatca tttgttatag gcatatcatt 1680

gatcaatgat agtaactcac tgtttgttaa catgtaagtg cttaaaggtg ttgttacacc 1740

tgcattgaca ctgaattctc tgttgatttc caacaatctg ctgttcttct gctggaattc 1800

tataactgtt tcaatgttgg agatgcgacg gttctgcctt tggtttgtgc tgttgtgtgg 1860

tgtgtttctg actttgtgtt gggtgatgtt gtggctgaat ttgtgtggat tggtgatgtg 1920

gttgtaggtt gtttggatga gctaaccctt tctggtggga cttgagtaag gtaggtggtg 1980

atgttttttt cagtgtggtt ttttattgtt tgaactgtga ccgttgttag tgtaactttg 2040

tgattggcag agatgatgaa tattatggct gcaattatga gagaggttga gattatcatt 2100

gccagaactg atagtgctat ttgtgctata gattttaaat ttaatctgta taaacaagag 2160

gatattacaa ttagatgatt gagagtatcc caggtctttt ctagagtcct ggcagtgcgt 2220

tgattcttgt gtttggacat ggttgcattt gccccaatgt tgtttttgat cccatactaa 2280

taattcatca ttatgttaat ttttaaataa ctactctatg ctaccatgat agttggcgtg 2340

gttttgcgac accatgactc tgtgagaaga ttggatttgt acatgaggat cacccatgtt 2400

taattaagg 2409

<210> 7

<211> 1757

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cggggtacca catgaggatc acccatgttt aaaactgggc tcgggttgta cccacccgag 60

ctgcccatgt ggcgtggtgc tcttgtatcc cggtacactt gcacgccagt ttgcctctcc 120

ttaccccgta acatatctag aagaatgtgc ataacggacc aataggtggt ggcaacccat 180

gccactgacg gtcaagtact tctgtttccc cggtgtgatg tggaatagac tctctcaggg 240

ttgaagccat aagcatcgtt atccgctacg tgcctacgca aaacctagta gcattttgaa 300

gcttacttgg ttggccgctc agccactaac ccgtggtagg cctggcagat gaggctgggc 360

gcaccccact ggcgacagtg gcccagcctg cgtggctgcc tgcccctctt tgaggggaag 420

ccatttaagt gacaaggtgt gaagagtctc gtgtgctcca cgtagaatcc ttccggcccc 480

tgaatgtggc taatcctaac cccgcagcta ttgcacacaa tccagtgtgt tgatagtcgt 540

aatgggcaac tgtgggatgg aaccaactac tttgggtgtc cgtgtttcct gttttcttta 600

aatacttgct tatggtgaca aatagtgtat actgtttgac accatgggcg cacaggtcag 660

caagcaaaat gtcggctcgc atgaaaactc agtctcagcc acaggtggat ccgtgattaa 720

gtatttcaac atcaattact acaaggattc tgctagctct ggcttgacta aacaagattt 780

ttcccaagac ccatcgaaat tcacacaacc tctagcagaa gcacttacaa atccagcttt 840

aatgtcacca actgttgaag catgtgggat gtccgatagg cttaaacaaa ttactatcgg 900

gaattccact ataacaacac aagatacact aaactctata ctggcatatg gggagtggcc 960

caaatacttg agtgacctgg acgcttcctc agtggataaa cctacccacc cagaaacatc 1020

atctgataga ttttacacat taactagtgt agattggacc actacgtcta aaggttggtg 1080

gtggaagttg cctgattgcc ttaaagatat gggcatcttc gggcaaaatc tgtaccatca 1140

tgcattgggt aggtcagggt acataataca cacccaatgt aatgccacaa aattcaatag 1200

tggttgtcta atagtggctg ttgtaccaga acaccagcta gcttacatag gtgaagcaaa 1260

tgtcaatgtt ggttatgatc acacacaccc tggtgaggga ggacatgtaa ttggttcaaa 1320

tgttaggaga gataacaagc aacctgatga agaccccttc tttaattgta atgggaccct 1380

gcttggtaac atcactatat tcccacacca gctcataaac ttgaggacaa acaattccag 1440

cacaattgtt gtaccataca ttaattgtgt acctatggac aacatgctca ggcacaacaa 1500

cctatctcta gttattattc caatcgttcc tctcagagcc gcaaatggtg tcaccaaggt 1560

ccccattaca atctcaatag caccagataa gtcagagttc tcaggggcta gacagtctgt 1620

aaaacagggg ttaccaacac ggctgccaag tggatctcaa caatttctca caactgagga 1680

cgagcagtcg gctaatcttc taccagactt tagtgtcaca aagatcatac atgaggatca 1740

cccatgttta attaagg 1757

<210> 8

<211> 2278

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cggggtacca catgaggatc acccatgttt tttcttattc tatttgtcat ataaatgtct 60

attcatgctt tgtttttaaa tttttataca tcatatctgg ttttcacata attgatatag 120

tgttgggtct tgatctgctc aagatgtgat tttacatatt ttagggatac ttgtcttgaa 180

caacataggt tgtaaggtat taaggctggc ttggttgatt tcaacaatgt ggaagcagta 240

gcccttcccg aaatgagtga tacatgatgt agtagtgtat gctgcctcta gttgtacatt 300

cttgagtctt agcatgttga taatttctga ggtatttgag tacattatgg tgggattaac 360

acgtgatgtg tttgcgtaca gtgtggttgt agcaacattg actgcatcag gagatagtgg 420

atatgcatca gtatatacac ctgatatgca ttctctcgga catctgttgt accagttgca 480

gtcttggttt cctggtcgag acaggacttc atgaggcgcc catttaatgg tcatggggtt 540

gttgatatct aatgatccta tttgcaggtt ggagtgccaa cctgaagatc tagtatatat 600

gtagatcttt ttacctagtt taagtagcct accttcggca cctaagtaat tttgagttat 660

tggaattgtc tcgacaacaa tctttggcct atcagataaa taattattga tacgaattaa 720

gacattgaca acttgtcttt tctttagcca agttatctta agagcatcat tgcaaacact 780

ctgattaaca ttggtacatc tgtttatcac acacttagtg tcgccttgga gcggagttgt 840

taagccaccg taccctagga aaatgagtgt attttcaatt tttatcccac ttcctacact 900

cggatacatt gcagaaaaag gatggtcaaa agttatatct tcatttttgt atcgatgaga 960

tttggtcttt cccttgagat ctaatatgtc aaatactaaa tcttctatac cttcactcga 1020

gtagtctgta gtctcattca cagtgggcaa ggagcataac tgataacccc ttgttcctgc 1080

agctattaca gaacatgatt tcctgttgtc gttgatgtca taggtatgag aaattaccgg 1140

gtttaaatca ggatacatat ctgaatttaa ggatatgtaa cctaattgta aaacctgata 1200

tgacttccct atatctgcac atccttgagt gattaagttt gatgaatacg catatattgc 1260

atcaccaatt gataatgaag gtagtctaac acatcctgaa attgtggtgg atccagaaag 1320

tagacttggt ccaggtaata atgagatatt ggggttgttg ctcagtaggg gttcccctac 1380

gggacatctc cagaaatcgt gtgggtccag aggagatatg ccgtctgcat ggtgaatagc 1440

aatggtgttt tcgcatatct gagccaattc ctgtctgttg catgacttct ctattaattg 1500

tgtgagatct ctgctttgct tgtttaacaa tattgggatc ccgctttgta ctgaactttg 1560

tatgtttatg gtccttgata ttacttcttg tctgattaat tctgtgattg tctctttgat 1620

tattttggca ctttcgttca tggaggatac tgtcatgatg tttgtcttca tacaagtgtc 1680

ttgttttata attaggtcaa tgcataggat catgataatg aaggacaata ctgtatgcat 1740

tgttgttgca atcagtagcc agatgtgtgt ccttcctgct ggtgtattaa tgtgtgtgtt 1800

taccgtggaa ttgtcatttc gggttgtaga ccaatatgaa ctatttgttt tccctttttc 1860

agccatcgtt tgtataatcg gatgctgttg ccttataggt tgactggatt gtctttaacc 1920

ctaagttttt cttacttgtt gtacacctga ttggtgaagt tgtggataga tttgattgtt 1980

gtatgtatgc tacaatgtac ttgatctgat tttttaattg gagttgttac ccatgtaggg 2040

atttctcatt cttgactcca gagtataagc attaacaggt gaattatgag ttgaattaat 2100

cattactaat agtcttctaa ccctgtatag atagtataat ataccacata tgattattat 2160

aagtatgcac acaattatta tcatgattat ctgagtactc tctgtgttgt gccatcctcc 2220

aactgctgat atgattgccc ttgccttcaa catgaggatc acccatgttt aattaagg 2278

<210> 9

<211> 2110

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cggggtacca catgaggatc acccatgtga tacatttctg tagaggctgg gttagatgtt 60

ttcaacacta ttaagctgac atctctccag tatcttaaat acaaacttaa ttgttttgtc 120

cagtctgtac ctaatcttgg tgcaattttg cttactaaga taacgtcctt atccccaaca 180

aggtatgcaa tcctgatcac actataatgt tcatgtaaga ccacctgatc acatttgtgt 240

tctccacctt ccatgtcaca atgaataaac cctattgaat tattctgtaa ttcattccag 300

attagtgttt cacattcatc attccctatc caagttgatc caggattccc attgaataaa 360

accttaactc tttgacataa actcgtgaca ttattcaatt tcttccctac cagtgccact 420

tctgaaggat aaatatttaa ttctctttgt ccgttgagat cacaagaatt aacacctgaa 480

ttataatagt tcatgcaggg tcctaatgta gcatcataac aagacagcat tgcacctgca 540

ccttctccga gatataatct atctttatcc ttattgacta gaggattcag taaataggta 600

agttccaatg ctttcaaaca actggtgcta ttaatcccga aaagtctcaa ttggtgtgat 660

aaatatcgcc catcaaaagg tagagttaat cctacacctg atcttcctct ttcttccctt 720

tttatctctc aatccggtta acataatttg ttgataaact aaattagtat ctttagactc 780

tcctgcctct tttaatgcca tattatcatt agatatggta ataaatcgac tcgctcgaac 840

taaagtagca cttgaaaatt tcatctgtgt agcagtatct ctcaatctgt gggacaaatt 900

tgtagaagtc gatacagggg tgagtaactt cctctcctga cactagctct tactagagat 960

ttcgttgtat caagcatccc ggctatagct tctcttacac ctgtaagtga gttatctaag 1020

atctcgtgag ctactctggg caatatggct ttcctatcca tcaaaaatga tgctaagttg 1080

agatcttctt cactacttga ttctgaaaag agacctgata ggagaggatt aggtgattcc 1140

tgcaatactg atctagctgt tacatttttg attatagttg ttatactttg tgagtgtggg 1200

agattgcatg aataagggtc tgatgcccaa tctaagaagc ttgagtctcc tggttcttga 1260

ttcatcacac gatataatac ctgtttatct aacagacctg ctttgatgaa tctctttaag 1320

tctgctagag ctgcaactgc tggatcacct atatttctga caaaacatct agctgtagac 1380

atatagttga accctcctat gttagctggg atcaatattg cacatcttaa ccaatttttc 1440

cctttaaaat attgatcttt gatagttgac gtaatagtac tgtaatttcc tacaatctga 1500

atgttcttct ctaatgtagt taaatctgac ctggatgtgt tcatagtcac cagagtttct 1560

accataagag atactaagct ttgttgtgac ctcatcactg caccaatacc ttcaactgtg 1620

tctcctgtga agaccagggc acttttaacg gttccatcct gtctgaatgc ttctaatcta 1680

ttgaagaatc cttttcttag accagcacta cttgttatgg ctttaaccaa cactatccaa 1740

acttgaacta ttatagctcc gagacatgca ggatatccat atgtctgaag caatgcctct 1800

agatccgcat tctctctttg cccttggaac aatgggttct tgttgatcat aggtccaaac 1860

aaccactctg ttgttctttc aaattgctcc tccgccagac ttattgatgc tctcacttct 1920

cctggaacta aatgtgtcaa aagtacttag tagcccggcc attataaaaa tttatactaa 1980

gatcaaatgt ctgtgacttg tccctttaaa gtattacttt aaccctaaaa tactatttaa 2040

tattattata tattccaaac aagtttttcc tcttgtttgg tacatgagga tcacccatgt 2100

ttaattaagg 2110

<210> 10

<211> 1789

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ccggccggcc acatgaggat cacccatgtt tatgattgac tgcagctttt tggaatctct 60

gttttgaaca acataggttg aaatgtgttt gaacttttct gatttatttc tactatatga 120

aaacaatatc ctttgtcata gtgtgtaatg cagcttgttg ttgtatatcc agctgagagt 180

gttctgtttc tgatggccag ctcgtttact ctttcagttg ctgttgagta agttatgact 240

gggttcactc tcgatttctg tgagtctagt atgacagatg acacaatgct ccctgtggga 300

tttagtggat acgcatcagt atatactcct gttatacatc catttggaca tgaatgtccc 360

catggacatt catcgtttcc tggtcttgat agcacattat gccatgtcca ttttatcctt 420

atatcactgt aatcagtaat atcaattatt cctaattgta atttgctatg ccaacttgta 480

gatcttgtat atatatagat cttgttacct agtagaatca accttccttc tgacccccag 540

taattttgtc tcatagatat tgtccatacc tttaattttg gaattgagtt taagcctttg 600

tcaacaacaa tgatggagtt gaccatcctc ctatctgaaa accatggact ataagatgcc 660

tgattgcagt ctctctgtgt tttcccggga cactcagttg tgttgcagat tacattctca 720

tttattggat gttcaagacc tccatacccg agaaatatta ttttgccttt gtaatatatc 780

cctggtccaa cagatgggta tagtgcagca tagggttgat caaagcttat gttattattc 840

ttaaatcttg ttgttgagat tgagccatca taattgacaa tatcaagtac aatatcttct 900

atgcctgatg atgcataatc tgatctttca tcaactttgg gagttgaaca cagttgatat 960

acatctgtat ttaggagtgc tagagaacat gacttcctat tgtcatttat gttgaaagta 1020

tgagagaacc tgggatttaa gtcaggtacc aagtctgagt ttacagttat tatccctacc 1080

tgtaagactt ggtatgattt tcctatatcc tgacaacctc gagtaattag atttgaggta 1140

taagcataaa tcagatcatt tataattaag gacggagttc tgacacagcc atcaacagtc 1200

gttggcatgg ctaataatcc cggccctggc attaacctta tttttgggtt tctcattaaa 1260

aatggaagac cagacgtgca cctccaaaaa tcatctggat ttaaaggttt tatacctaca 1320

tcatgtgtta ttctttgttg cggcacttct tgattatcat ttctaattgt aatttcacta 1380

atgaatttcc taagatccga catctgttgt gtcaatgata ttggtatata attctggaca 1440

tgactctgaa ttgtaagaag ccttgtgttc actcctgact gtattagatc attggtatta 1500

tccgatgcca tttggatctt ttctgtaatc tccataaact cattatttat ttcttgtagc 1560

aatgaattat ggaccttttc actattgatg gaattaatta gcactatgat gaaaactatt 1620

gataataaca ccaggattat tgtccataat atatatatta tcttattagt gagcttgttg 1680

ttatgagtag ccatggacgt ctccagctca ttaccagcat cctttccgtg attggtatgc 1740

ttccagtatt ccatctcgaa acatgaggat cacccatgtt taattaagg 1789

<210> 11

<211> 2240

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cggggtacca catgaggatc acccatgtta atgattttgt cgtatctaac attccagcta 60

tagcatttct aattcctgtg agagaattat ctagaatatc atgtgcaact ctagggagaa 120

ttaccttcct gtccatcagg aactcagcta attcttcatc ttcctctatc attgtatttg 180

tgaataatcc agataataat ggatttggtg aatcttgtaa tacattcctt gctgttatat 240

tttttatcat ggtggttata ttttgagatt gtggtaaatt gcatgaatat gggtctgaag 300

cccagtccaa aaaagatgac tcacctggtt cttggttcat aatcctataa agaacacttc 360

ggtctaatag attcgccttg ataaatcttt taatatcagc taatgcagca actgatggat 420

caccaatatt ccttacaaaa catcttgaca tagccatgta attgaatccc ccaacactgg 480

caggtattaa agaggcatat tgcatccaat ttgaattctt aaaatactga tctttgatat 540

tctgtgttat agttggattg atattcatcc caagggcaat atatagttgt tgaatattct 600

taaaaattga gcatgcatat cctagaacag gtgaataacc attctcaatt gcttttgcaa 660

atgatgttgc caagtttgaa gatgctgatc ttgtttcgtc tattactgtc tctgaccaaa 720

agacacatct agataatgct ttcagagctt gaggaagaat tctcccatca taatagattc 780

ttttgctata tatgaacatc ttgctactta taatcgtttc atttaattta agttcatgac 840

ctagatcatc catcacctct cttaatgaat caaaaaatct cactacatct ttataaacta 900

tctccttctt aattctgtaa tcataattgt ttggtactct tgtggttaca gctatagctt 960

gattgtctcc ttgaaccatt gcagtcaccc tcacgcctat tctaacagct gctagatgta 1020

ttgcacttat agatatgagt gtccataatt tttgacaaaa tccttctata ccccctcttg 1080

ggttatgaac ataaaaacca gaatcagggt gatcctctaa tgatatatgt tctttatctg 1140

atggagggca gtaaggatca cctacataga ttgtacttcc ttcaagacga gggtgtaacc 1200

aattaaacaa tttatttaat ccaaatattt ggttgcaagt ttctccaaat agagctgttg 1260

attcatatct ccaattaaga cagtattttt tgagatctgt tgttaggaaa cagctcacag 1320

tctcatatcc atcattgtag atatccgttg acttgaattc aaatttctta gatttctgat 1380

tggaagacaa attaagatta cttattttat tgtaggtttt aaggtcatct gtatggcttt 1440

tagaattatt gtacacttca ttataccgtg gaactcctga tattgatatg gtcgttaatc 1500

tcttaagtaa ttcaatctct cccttcacca tcccattctc ttgaaagaag atattgtgtt 1560

gggtagagat aatgatgcca ttgacttagg aattttgaac aaggttattg atccaattgc 1620

cgtacaattc acgaagatta ctctaaattt tatgctccta tctagtggaa gacattgagg 1680

agcaagagca actttgttgg catcgaacag cattcctttt cttagtctat tagaccatgg 1740

gtatagttct ggttttatat tttgtactgt atacacaacc atctctttcg ctttgactgt 1800

tcttctcact ggcaggatag ttgcctggtg cgaactcacc atgtatggga tctctgagga 1860

tacagatgaa aggagcgcgt ggtccctttg ataaatacag ttccatcaaa gcttttaatc 1920

tattgatgtc tggtctgaga gtggatagag tcaaagctgc cattctagtc tcaattccat 1980

atctgattgt attgaagaat gaagcgagac catttttctg tccaggaatg atagctccac 2040

cggctgattt tgttatgttt tcttgcctac gtgcattaaa tgtatcaaat aggctcaaca 2100

tttttgaaat tatagagttc ccttttcttg accttctagt caatgtcttt aatcctaagt 2160

taattttaat ttaaatttat atttccaaac aagtctcttc tcttgtttgg tacatgagga 2220

tcacccatgt ttaattaagg 2240

<210> 12

<211> 1959

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ccggccggcc acatgaggat cacccatgtt taaaacagcc tgtgggttgt acccacccac 60

agggcccact gggcgctagc actctgattc tacggaatcc ttgtgcgcct gttttatgtc 120

ccttccccca atcagtaact tagaagcatt gcacctcttt cgaccgttag caggcgtggc 180

gcaccagcca tgtcttggtc aagcacttct gtttccccgg accgagtatc aatagactgc 240

tcacgcggtt gagggagaaa acgtccgtta cccggctaac tacttcgaga agcctagtag 300

caccatgaaa gttgcagagt gtttcgctca gcacttcccc cgtgtagatc aggtcgatga 360

gtcactgcga tccccacggg cgaccgtggc agtggctgcg ttggcggcct gcctgtgggg 420

taacccacag gacgctctaa tatggacatg gtgcaaagag tctattgagc tagttagtag 480

tcctccggcc cctgaatgcg gctaatccta actgcggagc acataccctc gacccagggg 540

gcagtgtgtc gtaacgggca actctgcagc ggaaccgact actttgggtg tccgtgtttc 600

cttttattct tatactggct gcttatggtg acaattgaaa gattgttacc atatagctat 660

tggattggcc atccggtgtg caacagagct attatttacc tatttgttgg gtatatacca 720

ctcacatcca gaaaaaccct cgacacacta gtatacattc tttacttgaa ttctagaaaa 780

cagcctatat tgtggccctc gcagccgctc aggacaactt taccatgaaa ctgtgcaaag 840

acactgagga tattgagcaa tctgcaaaca tccagggtga tggaattgca gacatgattg 900

accaggctgt cacttcccga gttggtcgtg cgctgacatc cttacaggta gaacctaccg 960

ccgccaacac caatgctagt gagcacagat tgggcaccgg gctcgtcccc gccttgcagg 1020

ctgcagagac cggcgcctct tctaatgcac aggatgagaa tcttatagaa acccggtgtg 1080

tgttgaacca tcactccact caagagacca cgattggcaa ctttttcagt cgagcaggac 1140

tagtgagtat tattaccatg cccaccacag gtacccaaaa caccgatggg tatgtgaact 1200

gggatattga cttgatgggt tatgctcaaa tgaggcgtaa gtgtgagcta ttcacataca 1260

tgcgctttga tgcagagttt acatttgtag ctgccaaacc aaacggtgag ctagtaccac 1320

aattgttgca gtacatgtat gtgcctcccg gagctccaaa acctacgtcc cgggattcct 1380

ttgcctggca gactgctacc aatccttcca tcttcgtcaa gttgactgac cccccggcac 1440

aagtgtcagt acccttcatg tctcccgcca gcgcctacca gtggttttac gatggctatc 1500

caacgtttgg agcccatcca caatcgaatg acgcagacta tggccaatgt ccaaacaaca 1560

tgatgggcac ctttagcatc agaactgtag gcactgagaa atccccccat tctatcaccc 1620

ttcgggtgta tatgagaatc aagcatgtca gggcctggat accccggcca ctcagaaacc 1680

aaccgtacct ttttaagaca aatccaaatt ataaaggcaa cgacatcaaa tgcaccagca 1740

caagtaggga caaaataaca acactaggaa aatttggcca gcaatctggt gctatttacg 1800

tgggtaacta tagagtggtc aatcgtcact tggccacgca caatgactgg gccaatttag 1860

tatgggaaga cagctcaagg gatcttttag tctcctccac cactgcacag ggatgtgaca 1920

ctattgctcg acatgaggat cacccatgtt taattaagg 1959

<210> 13

<211> 1859

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ccggccggcc acatgaggat cacccatgtt taaaacagct gtgggttgtc acccacccac 60

agggtccact gggcgctagt acactggtat ctcggtacct ttgtacgcct gttttatacc 120

ccctccctga tttgcaactt agaagcaacg caaaccagat caatagtagg tgtgacatac 180

cagtcgcatc ttgatcaagc acttctgtat ccccggaccg agtatcaata gactgtgcac 240

acggttgaag gagaaaacgt ccgttacccg gctaactact tcgagaagcc tagtaacgcc 300

attgaagttg cagagtgttt cgctcagcac tccccccgtg tagatcaggt cgatgagtca 360

ccgcattccc cacgggcgac cgtggcggtg gctgcgttgg cggcctgcct atggggtaac 420

ccataggacg ctctaatacg gacatggcgt gaagagtcta ttgagctagt tagtagtcct 480

ccggcccctg aatgcggcta atcctaactg cggagcacat acccttaatc caaagggcag 540

tgtgtcgtaa cgggcaactc tgcagcggaa ccgactactt tgggtgtccg tgtttctttt 600

tattcttgta ttggctgctt atggtgacaa ttaaagaatt gttaccatat agctattgga 660

ttggccatcc agtgtcaaac agagctattg tatatctctt tgttggattc acacctctca 720

ctcttgaaac gttacacacc ctcaattaca ttatactgct gaacacgaag cgatgggcta 780

aggacaccag tgacattttg gaaacggcca ctattcaagg ggacagagtg gcagatgtga 840

ttgagagctc tataggagat agtgtgagta aggccctcac cccagcttta cctgcaccca 900

caggcccaga cacccaagtg agcagtcatc gcttagacac tggaaaagta ccagcacttc 960

aagccgccga aatcggagct tcgtcgaatg ctagtgatga gagtatgatt gagactcggt 1020

gtgttcttaa ctcacatagc acagctgaaa ccacccttga tagtttcttc agcagagcag 1080

gcttagttgg ggagatagat cttcctctaa agggcaccac caatccgaac gggtatgcca 1140

actgggacat agacataacc ggttatgcgc agatgcgcag aaaagtggaa ctattcacct 1200

atatgcgctt tgacgcagag ttcacttttg tcgcgtgcac acctaccgga agggtcgttc 1260

cacagctgct tcaatacatg tttgttccac ccggggcccc caaaccagac tccagagact 1320

ctttggcttg gccaacggcc acgaacccct cagtttttgt caaatcatcc gacccaccag 1380

cacaagtctc agtgccattt atgtcacctg caagcgcata ccaatggttt tatgacggat 1440

accctacatt tggagagcac aagcaagaga aggatctcga gtatggggca tgcccgaata 1500

acatgatggg cacattctca gtgcggactg tgggatcgtc aaagtcagaa tattccttag 1560

tcatcagaat atacatgaga atgaagcacg tcagagcgtg gatacctcgg ccgatgcgca 1620

atcagaacta tttgttcaaa tccaacccaa actatgctgg tgattccatt aaaccaactg 1680

gtaccagccg aacggcaatc actacgctcg ggaaattcgg tcagcagtct ggggctattt 1740

atgtgggcaa ctttagggta gtaaacagac acctagccac ccatactgac tgggccaact 1800

tggtgtggga agacagctct agagacctcc acatgaggat cacccatgtt taattaagg 1859

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gggcatattc ataaacctca 20

<210> 15

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tacccaagga catagccaa 19

<210> 16

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tccccaccgt aacatcact 19

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

agttacacct gcattaacac 20

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cagccatcat attcatagcc 20

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

aagccagatc aagaacacaa cc 22

<210> 20

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tctatactct cccattatgc cta 23

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tgtttatgaa tgcctatggt 20

<210> 22

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ctaagatggg gagttttagc caa 23

<210> 23

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

aatgtcttta taatcccacg a 21

<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

accttgctgg aaattacaca 20

<210> 25

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

cctgcattat cacaatacca tcc 23

<210> 26

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ctgaatgtgg ctaatcctaa ccc 23

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

aaaacaggaa acacggacac 20

<210> 28

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

atcccacagt tgcccattac gac 23

<210> 29

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

attattttgg cactttcgt 19

<210> 30

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

agtcaaccta taaggcaaca 20

<210> 31

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ttgtcatttc gggttgtaga cca 23

<210> 32

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

atgggcgata tttatcaca 19

<210> 33

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

tttaattctc tttgtccgtt 20

<210> 34

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

acttttcggg attaatagca c 21

<210> 35

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

ttttgccttt gtaatatatc cc 22

<210> 36

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ttattagcca tgccaacga 19

<210> 37

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

ttaaggacgg agttctgaca c 21

<210> 38

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

ggtctaatag attcgcctt 19

<210> 39

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

aatatgcctc tttaatacct g 21

<210> 40

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

atagccatgt aattgaatcc cc 22

<210> 41

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

aaacggtgag ctagtaccac aa 22

<210> 42

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ccatagtctg cgtcattcga tt 22

<210> 43

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

cccggcacaa gtgtcagtac cc 22

<210> 44

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

agtgagcagt catcgcttag aca 23

<210> 45

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

atctatctcc ccaactaagc ct 22

<210> 46

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

ccagcacttc aagccgccga aa 22

Claims (10)

1. The quality control product for nucleic acid detection of seven RNA viruses is characterized in that: has a nucleotide sequence shown in a sequence table SEQ ID NO.3-SEQ ID NO.13, wherein:

(1) the RNA virus is respiratory syncytial virus A type virus and has nucleotide sequences shown in a sequence table SEQ ID NO.3 and a sequence table SEQ ID NO. 4;

(2) respiratory syncytial virus B type virus, having nucleotide sequences shown in sequence tables SEQ ID NO.5 and SEQ ID NO. 6;

(3) rhinovirus C type virus with nucleotide sequence shown in sequence table SEQ ID NO. 7;

(4) parainfluenza virus type 1 having a nucleotide sequence represented by SEQ ID NO.8 or SEQ ID NO.9 of the sequence Listing;

(5) parainfluenza virus type 3 having a nucleotide sequence represented by SEQ ID NO.10 or SEQ ID NO.11 of the sequence Listing;

(6) coxsackievirus A16 type virus, having a nucleotide sequence shown in a sequence table SEQ ID NO. 12;

(7) the enterovirus71 has a nucleotide sequence shown in a sequence table SEQ ID NO. 13.

2. The nucleic acid detection quality control product according to claim 1, wherein: the seven RNA virus nucleic acid detection quality control products are MS2 virus-like particle nucleic acid detection quality control products of seven RNA viruses.

3. The nucleic acid detection quality control product according to claim 1 or 2, which is prepared by the following method:

1) constructing a double-expression system vector plasmid pACYC-MS2-virus simultaneously containing an MS2 bacteriophage gene sequence and a virus recombinant conventional detection region to obtain a recombinant vector;

2) transforming BL21(DE3) E.coli, IPTG induced expression and purifying MS2 virus-like particles by using sephadex gel filtration chromatography;

3) packaging the virus-like particles by MS2 and verifying the sequence correctness by adopting a series of methods of nuclease digestion, polyacrylamide gel electrophoresis, reverse transcription PCR, Sanger sequencing and real-time fluorescence reverse transcription PCR; absolute quantification is carried out by adopting liquid drop digital PCR;

4) diluting and subpackaging a sample by adopting DMEM diluent; lyophilizing with Christ ALPHA 1-2 type lyophilizer at 0.37mbar for 25 hr to obtain the final product.

4. The preparation method of the seven RNA virus nucleic acid detection quality control products of claim 1 or 2, which is characterized by comprising the following steps:

1) constructing a double-expression system vector plasmid pACYC-MS2-virus simultaneously containing an MS2 bacteriophage gene sequence and a virus recombinant conventional detection region to obtain a recombinant vector;

2) transforming BL21(DE3) E.coli, IPTG induced expression and purifying MS2 virus-like particles by using sephadex gel filtration chromatography;

3) packaging the virus-like particles by MS2 and verifying the sequence correctness by adopting a series of methods of nuclease digestion, polyacrylamide gel electrophoresis, reverse transcription PCR, Sanger sequencing and real-time fluorescence reverse transcription PCR; absolute quantification is carried out by adopting liquid drop digital PCR;

4) diluting and subpackaging a sample by adopting DMEM diluent; lyophilizing with Christ ALPHA 1-2 type lyophilizer at 0.37mbar for 25 hr to obtain the final product.

5. The method of claim 4, wherein: the double expression system carrier plasmid pACYC-MS2-virus takes pACYCDuet-1 plasmid as a carrier, capsid protein gene and mature enzyme gene sequences of MS2 bacteriophage are respectively inserted into a multiple cloning site 1 of the plasmid in a restriction enzyme cutting mode, a recombinant conventional detection region sequence of related viruses is inserted into a multiple cloning site 2 of the plasmid, and the recombinant carrier plasmid can simultaneously express MS2 capsid protein and exogenous virus RNA.

6. The method of claim 4, wherein: the recombinant conventional detection region sequence covers the target region of most of the existing detection methods and detection kits, and the two ends of the recombinant sequence are connected with an optimized MS2 phage packaging signal and an optimized enzyme digestion site.

7. The method of claim 4, wherein: the real-time fluorescent reverse transcription PCR and liquid drop digital PCR quantitative method adopts a primer probe sequence which is a nucleotide sequence shown in a sequence table SEQ ID NO.14-SEQ ID NO. 46.

8. Use of the seven RNA virus nucleic acid detection quality control products of claims 1 to 3 in the preparation of laboratory quality control reagents.

9. A quality control reagent for RNA virus nucleic acid detection is characterized in that: contains seven RNA virus nucleic acid detection quality control products, which have nucleotide sequences shown in sequence tables SEQ ID NO.3-SEQ ID NO.13 and are MS2 virus-like particle nucleic acid detection quality control products of seven RNA viruses.

10. The quality control reagent according to claim 9, wherein: further comprises primer probe sequences which have nucleotide sequences shown in sequence tables SEQ ID NO.14-SEQ ID NO. 46.

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