CN108866238B

CN108866238B - Kit for detecting various insect-borne viruses and invertebrate infectious viruses

Info

Publication number: CN108866238B
Application number: CN201710345060.0A
Authority: CN
Inventors: 谢良志; 孟淑芳; 孙春昀; 刘茜; 吴雪伶; 徐苗; 李久凤; 樊金萍; 王保; 赵翔; 冯建平
Original assignee: Beijing Yiqiao Shenzhou Polytron Technologies Inc; National Institutes for Food and Drug Control
Current assignee: Beijing Yiqiao Shenzhou Polytron Technologies Inc; National Institutes for Food and Drug Control
Priority date: 2017-05-16
Filing date: 2017-05-16
Publication date: 2021-12-31
Anticipated expiration: 2037-05-16
Also published as: CN108866238A

Abstract

The invention provides a kit for detecting various arbovirus and invertebrate infectious viruses, belonging to the technical field of fluorescent PCR detection. The kit contains 35 pairs of specific primers aiming at 158 viruses of 11 invertebrate viroids and 5 arbovirus families, 35 positive plasmid standard substances, fluorescent PCR reaction liquid and sterile H₂O, the nucleotide sequences of the 35 pairs of specific primers are respectively shown as SEQ ID NO. 1-70; the nucleotide sequences of the positive plasmid standard products are respectively shown in SEQ ID NO. 71-105. The kit can rapidly detect various insect-borne viruses and invertebrate infectious viruses, has the advantages of simple operation, high sensitivity, good specificity, reliable and stable detection result and the like, can be used for the safety detection of insect viruses in an insect cell production system, improves the safety of medicines, and has good application prospect in the field of biological product production.

Description

Kit for detecting various insect-borne viruses and invertebrate infectious viruses

Technical Field

The invention relates to a kit for detecting various invertebrate infection viruses and arboviruses of insect cells by using a fluorescence-PCR reaction technology, belonging to the technical field of biological detection.

Background

Arboreal disease refers to a disease that is transmitted from blood-sucking arthropods to humans and humans, and to humans and animals by bite infections. The clinical manifestations of the animal are sudden fever, ache and weakness, large area of rash, death caused by secondary infection, etc. The disease causes thousands of people and livestock to be infected and even die in the past, and brings certain negative effects to the development of economy and human society. The continuous development of international animal trade has also been promoted in the middle of the 20 th century with the global increase in traffic, particularly with the use of modern vehicles that have shortened the distance around the world, these factors providing a wider possibility for the long-range spread of arboreal diseases and the tendency to expand the popularity worldwide. At present, with increasingly frequent international communications, the risk of mosquitoes, ticks, midges and other vectors being introduced into the border port of China is very high, and outbreaks of insect-borne animal diseases are also likely to be introduced into China along with biological insect vectors.

There have been many product attempts to use an insect cell production system as a Protein production platform, and among them, cervarix hpv (human papilloma virus) vaccine by GSK company and FluBlok influenza vaccine by Protein Science company have been marketed abroad. The safety of an insect cell production system is monitored, and an insect cell exogenous virus quality control system is established, so that the method is one of important methods for controlling the safety of medicaments.

Unlike the consideration of virus risk profile of mammalian cells, the evaluation of virus safety of insect cells must consider the specific virus risks of insects including invertebrate virus and arbovirus in addition to the conventional virus risks, such as the existing reports in the literature that the infection of nodavirus (nodaviruses) exists in HighFive cells and the infection of rhabdovirus (rhabdoviruses) exists in Sf9 cells, so that the establishment of the detection method of invertebrate virus and arbovirus is important for controlling the safety of drug production.

Invertebrate infectious viruses include: (1) ascoviridae vesiculoviridae, (2) Iridovirdae Iridoviridae, (3) Poxviridae, (4) Polydnaviridae, (5) Baculovirae Baculoviridae, (6) Parvoviridae Parvoviridae, (7) Birnaviridae binuclear uronoviridae, (8) Reoviridae Reoviridae, (9) Nodavridae Nodaviridae, (10) Picornaviruses picornaviridae (Dicystroviridae bicistronic viridae), (11) Teviridae. Arboviruses include: (1) reoviridae (orbivirus) reoviridae (circoviridae), (2) togavirus togaviridae, (3) flavivirus flaviviridae, (4) bunyavirus bunyaviridae, (5) asfarvirus swine fever virus.

Fluorescence PCR is a molecular biology technique that directly detects nucleic acids and allows quantification. Has the advantages of good specificity, high sensitivity, good accuracy, simple operation, pollution prevention and the like. It is a method for measuring the total amount of products after each PCR cycle by adding a fluorescent group in the DNA amplification reaction and using a fluorescent chemical substance. The invention adopts different fluorescent PCR methods to carry out amplification detection on sample nucleic acid so as to identify whether the sample is infected by the arbovirus and the invertebrate virus.

Disclosure of Invention

The invention aims to provide a kit for detecting insect cells and the pollution of arbovirus and invertebrate virus in samples prepared from the insect cells by using fluorescence PCR, and whether the samples are infected by the arbovirus and the invertebrate virus can be quickly and accurately detected by using the kit.

The invention firstly provides a specific primer pair combination for detecting the arbovirus and the invertebrate infectious virus, which comprises the following steps:

(1) the nucleotide sequence of the primer pair for detecting Reoviridae Bluetongue virus family is shown as SEQ ID NO 1-2;

(2) the nucleotide sequence of the primer pair for detecting the togaviruses togaviridae is shown as SEQ ID NO. 3-4;

(3) the nucleotide sequence of the primer pair for detecting flaviviruses is shown as SEQ ID NO. 5-6;

(4) the nucleotide sequence of the primer pair for detecting the Bunyavirus bunyaviridae is shown as SEQ ID NO 7-10;

(5) the nucleotide sequence of the primer pair for detecting the asfarvirus classical swine fever virus family is shown as SEQ ID NO. 11-12;

(6) the nucleotide sequences of the primer pairs for detecting the Ascoviaridae vesiculoviridae are shown as SEQ ID NO 13-14;

(7) the nucleotide sequence of the primer pair for detecting Iridoviridae Iridoviridae is shown as SEQ ID NO. 15-22;

(8) the nucleotide sequences of the primer pairs for detecting Poxviridae Poxviridae are shown as SEQ ID NO 23-28;

(9) the nucleotide sequence of the primer pair for detecting Baculoviridae baculovirus is shown as SEQ ID NO. 29-32;

(10) the nucleotide sequences of the primer pairs for detecting the Polydnaviridae are shown as SEQ ID NO 33-36;

(11) the nucleotide sequences of the primer pair for detecting Parvoviridae Parvoviridae are shown as SEQ ID NO. 37-44;

(12) the nucleotide sequence of the primer pair for detecting the Birnaviridae binuclear glycoviridae is shown as SEQ ID NO. 45-46;

(13) the nucleotide sequences of the primer pair for detecting Reoviridae are shown as SEQ ID NO 47-52;

(14) the nucleotide sequence of the primer pair for detecting the Nodaviridae is shown as SEQ ID NO:53-58

(15) The nucleotide sequence of the primer pair for detecting Picornavirals picornavirus (Dicystroviridae bicistronic virus) family is shown as SEQ ID NO 59-64;

(16) the nucleotide sequence of the primer pair for detecting the Tetraviridae tetrahviridae is shown as SEQ ID NO 65-70;

the invention provides application of the specific primer pair combination in preparing a kit for detecting various arbovirus and invertebrate infectious viruses.

The invention provides a kit containing the specific primer pair combination.

The kit detects the virus types and the corresponding nucleic acid types as follows:

(1) the virus nucleic acid type of Reoviridae Bluetongue virus is dsRNA

(2) the viral nucleic acid type of togaviruses Togavirus is ssRNA (+)

(3) The viral nucleic acid type of flaviviruses is ssRNA (-)

(4) The viral nucleic acid type of Bunyavirus bunyaviridae is ssRNA (-)

(5) The virus nucleic acid type of asfarvirus, classical swine fever virus family is dsDNA

(6) The viral nucleic acid type of Ascoviaridae vesiculoviridae is dsDNA

(7) The virus nucleic acid type of Iridovirdae Iridoviridae is dsDNA

(8) The viral nucleic acid type of Poxviridae family is dsDNA

(9) The viral nucleic acid type of the Polydnaviridae family is dsDNA

(10) The virus nucleic acid type of Baculoviridae is dsDNA

(11) The viral nucleic acid type of Parvoviridae Parvoviridae is ssDNA

(12) The virus nucleic acid type of Birnaviridae binuclear glycoviridae is dsRNA

(13) The virus nucleic acid type of Reoviridae is dsRNA

(14) The viral nucleic acid type of the Nodaviridae family is ssRNA (+)

(15) The viral nucleic acid type of Picornavirales picornavirus (Dicystroviridae bicistronic virus) family is ssRNA (+)

(16) Viral nucleic acid type of Tetraviridae was ssRNA (+)

The kit contains 35 positive control plasmid standards, is used as a positive plasmid corresponding to a primer for detecting various arbovirus and invertebrate infectious viruses, and has the following sequence:

(1) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting reoviridae (orbiviruses) reoviridae is shown as SEQ ID NO. 71;

(2) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the togaviruses togaviridae is shown as SEQ ID NO: 72;

(3) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting flaviviruses is shown as SEQ ID NO. 73;

(4) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the Bunyavirus bunyaviridae is shown as SEQ ID NO 74-75;

(5) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the asfarvirus classical swine fever virus family is shown as SEQ ID NO. 76;

(6) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting Ascoviarie vesicoviridae is shown as SEQ ID NO: 77;

(7) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting Iridovirdae iridovirus is shown as SEQ ID NO. 78-81;

(8) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting Poxviridae Poxviridae is shown as SEQ ID NO 82-84;

(9) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the Polydnaviridae polygenetic DNA virus family is shown as SEQ ID NO 85-86;

(10) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting Baculoviridae is shown as SEQ ID NO. 87-88;

(11) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting Parvoviridae Parvoviridae is shown as SEQ ID NO. 89-92;

(12) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the Birnaviridae binuclear glycoviridae is shown as SEQ ID NO. 93;

(13) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting Reoviridae is shown as SEQ ID NO 94-96;

(14) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the Nodaviridae is shown as SEQ ID NO 97-99;

(15) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting Picornavirases picornavirus (Dicystroviridae bicistronic virus) family is shown as SEQ ID NO: 100-102;

(16) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the Tetraviridae four-virus family is shown as SEQ ID NO. 103-105;

the invention discloses a detection kit for various arbovirus and invertebrate infectious viruses, which comprises the following working procedures:

(1) extracting nucleic acid of a sample to be detected;

(2) the specific primer set according to claim 1, wherein the results are determined from an amplification curve and a melting curve by performing a fluorescence PCR reaction on each of the sample nucleic acids.

The adopted fluorescent PCR detection system comprises 10 mu L of fluorescent PCR reaction solution, 1 mu L of primer pair combination mixture, 5 mu L of to-be-detected product and sterile H₂O4. mu.L, 20. mu.L of the total reaction. In the fluorescence PCR detection system of the kit, the to-be-detected product is a sample nucleic acid extracting solution, a reverse transcription product of the sample nucleic acid extracting solution, a positive control plasmid or a negative control.

The 35 pairs of specific primer pairs contained in the kit are dry powder, each pair of primers is a mixture of an upstream primer and a downstream primer, and only 200 mu L of sterile water is needed to be added for dissolving the primers when the kit is used.

The positive plasmid reference substances of the invention are all packaged separately, and the plasmid concentrations are all 5 multiplied by 10⁸-5×10¹⁰copy/mL。

The preparation method of the sample nucleic acid extracting solution can refer to the conventional virus genome extraction technology. Depending on the type of virus, the fluorescent PCR assay is performed directly for DNA viruses, or for RNA viruses after reverse transcription of viral RNA into cDNA.

The fluorescent PCR reaction program of the kit is as follows: 5min at 95 ℃; reacting at 95 ℃ for 10s, at 60 ℃ for 20s and at 72 ℃ for 10s for 45 cycles; 5s at 95 ℃; 1min at 65 ℃; 65-97 deg.C (heating rate 0.11 deg.C/s); 30s at 40 ℃.

The use method of the kit comprises the following steps:

(1) preparing a sample nucleic acid extracting solution;

(2) preparation of reagents: diluting a positive standard substance and a primer, uniformly mixing and centrifuging a fluorescent PCR reaction solution;

(3) sample adding: preparing a detection mixed solution according to a fluorescent PCR detection system, placing the detection mixed solution into a PCR tube, and respectively placing a sample nucleic acid extracting solution, a positive control substance and a negative control substance into different PCR tubes of the reaction mixed solution to obtain a sample reaction tube, a negative control reaction tube and a positive control reaction tube;

(4) and (3) PCR amplification: the reaction tube is arranged on a fluorescent PCR instrument, and circulation parameters are set for PCR reaction;

(5) and after the PCR reaction is finished, detecting the fluorescence signal value and the melting temperature of the PCR reaction system.

The quality control of the kit comprises the following steps: each control of the kit must meet the requirements in table 1 below, otherwise the experiment is deemed invalid.

TABLE 1 kit quality standards

Control	CT value
		H₂O	N/A, 40 or melting temperature is a non-specific value
Positive control	≤35

The judgment criteria for the results of the sample nucleic acid extract are shown in Table 2:

TABLE 2 judgment standards for test results

Serial number	CT value	Tm value	Result judgment
				1	N/A or 40	Presence or absence of specific Tm value	Report as negative
2	<40	Having a specific Tm value	Report as positive
				3	<40	Non-specific Tm value	Report as negative

The invention provides application of the specific primer pair combination or the kit containing the 35 pairs of specific primers in monitoring the production safety of insect cells.

The invention provides application of the specific primer pair combination or the kit containing the 35 pairs of specific primers in controlling the safety of medicines in the field of medicine preparation.

The invention discloses 35 pairs of primers for detecting various arbovirus and invertebrate infectious viruses, a virus sequence amplified by the 35 pairs of primers, and a fluorescence PCR detection method for arbovirus and invertebrate infectious viruses. The kit has the advantages of simple operation, high sensitivity, good specificity and the like, and the detection result is reliable and stable, so that the kit can be applied to monitoring the safety of an insect cell production system and controlling the safety of drugs.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; all reagents used in the examples are commercially available unless otherwise specified.

PCR primer (Shanghai Czeri bioengineering, Inc.); LightCycler SYBR Green i Master (Roche, cat. No. 04887352001); TIANGEN plasmid mini-extraction kit (cat No. dp103); the QIAamp Viral RNA mini Kit (Qiagen,

cat No. 52904). Fluorescent PCR instrument (model: Lightcycler480II, manufacturer: Roche); vacuum centrifugal concentrating drier (cargo number LNG-T83B, Taicang).

EXAMPLE 1 design and Synthesis of primers for detection of various arboviruses and invertebrate infectious viruses

In order to cover more virus species and quantity of the same virus family to the maximum extent, more basic groups in the Primers can be designed into DEgenerate Primers, the specificity and sensitivity of amplification can be reduced by adopting the traditional DEgenerate Primers, the DEgenerate Primers of the invention all adopt a CODEHOP DEgenerate primer design method (Consensus-DEgenerate Hybrid Oligonucleotide Primers) to design virus amplification Primers (the DEgenerate primer numbering rule is V (A + C + G),

d (A + T + G), B (T + C + G), H (A + T + C), W (A + T), S (C + G), K (T + G), M (A + C), Y (C + T), R (A + G), N (A + G + C + T), I (deo xyInosine), U (deoxyUridine)). The method comprises the steps of firstly, obtaining a 3 ' degenerate core region of a 3-4 amino acid sequence (9-12 basic groups) design primer which is continuously conserved in a conserved region according to sequence comparison, and predicting an optimal pairing sequence to be designed into a 5 ' non-degenerate splint region according to the principles of amino acid conservation and codon preference in a 5 ' region. Although the 5 'specific region may be mismatched with the template sequence in the primary amplification, the primer will be disabled during the extension process due to the mismatch of the 3' core region, and the specific match of the primer will be greatly increased after several amplification rounds, thereby improving the specific

Accuracy and precision. The primer designed by the method has the advantages of long length, low degeneracy, high annealing temperature and

higher amplification specificity and sensitivity, and can be used for amplifying the same virus family

The virus is in full.

TABLE 3 primer degeneracy for arbovirus detection

TABLE 4 primer degeneracy for invertebrate Virus detection

The optimal primer sequences after screening are as follows:

(16) the nucleotide sequence of the primer pair for detecting the Tetraviridae tetrahviridae is shown as SEQ ID NO 65-70.

The sequences of the positive control plasmid standards are respectively as follows:

(1) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the bluetongue virus family is shown as SEQ ID NO: 71;

(2) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the togaviridae is shown as SEQ ID NO: 72;

(3) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the flaviviridae family is shown as SEQ ID NO: 73;

(4) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the bunyaviridae is shown as SEQ ID NO 74-75;

(5) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the hog cholera virus family is shown as SEQ ID NO. 76;

(6) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the vesicoviridae is shown as SEQ ID NO: 77;

(7) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting iridovirus is shown as SEQ ID NO: 78-81;

(8) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the poxviridae is shown as SEQ ID NO 82-84;

(9) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the multi-component DNA virus family is shown as SEQ ID NO. 85-86;

(10) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the baculovirus family is shown as SEQ ID NO 87-88;

(11) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting parvoviridae is shown as SEQ ID NO. 89-92;

(12) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the binuclear glycogenoviridae is shown as SEQ ID NO: 93;

(14) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the Rodaviridae is shown as SEQ ID NO: 97-99;

(15) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the picornaviridae is shown as SEQ ID NO 100-102;

(16) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the four virus families is shown as SEQ ID NO. 103-105.

EXAMPLE 2 preparation of Positive plasmid Standard of detection kit for various arbovirus and invertebrate infectious viruses

The synthesized positive plasmid was transformed into DH5a competent cells, plated and cultured overnight at 37 ℃. 4 monoclonal colonies were picked and transferred to 10mL LB liquid medium, cultured overnight and then extracted with a TIANGEN plasmid mini-extraction kit. Detecting the concentration of the plasmid standard substance by using an ultramicro ultraviolet spectrophotometer, calculating the plasmid concentration according to a plasmid concentration calculation formula (OD 260 multiplied by 50), sending the positive plasmid with qualified plasmid concentration to a sequencing company for sequencing, and preserving the seeds after the sequencing is correct.

Extraction of positive control plasmid: plasmid extraction was performed on the positive control plasmid following the procedure of the TIANGEN plasmid mini-extraction kit (cat. No. dp103).

Preparation method of positive plasmid standard substance

(1) Carrying out centrifugation on 13000 obtained positive control plasmid for 10 min;

(2) adding 30ul of protective agent into a 2ml storage tube, subpackaging the positive plasmids into 20ug per tube, subpackaging the plasmids, and slightly shaking the plates to uniformly mix the protective agent and the plasmids as much as possible.

(3) Plasmid extraction drying was performed according to the operating procedure of a vacuum centrifugal concentration dryer (cat No. LNG-T83B, Taicang).

(4) And (4) sending the dried plasmid to a sequencing company for sequencing identification, and storing at 4 ℃ after correct identification.

EXAMPLE 3 detection of multiple arbovirus and invertebrate infectious Virus detection kits primer sensitivity analysis

1. Preparation of samples

Taking 100 μ L of sterile H₂O was added to the aspirated plasmid standards at a concentration of 200 ng/. mu.L. According to plasmid copy number (6.02X 10)¹⁴The equation x plasmid concentration)/plasmid length/660 calculates the initial copy number of the plasmid standard. Plasmid standards were diluted to 5X 10 in a copy number 10-fold gradient⁷5copy/PCR reaction system, the sensitivity of the primer was initially determined by the fluorescent PCR method. Medicine for takingSensitivity and sensitivity the next gradient was subjected to fluorescent PCR using the corresponding primers, eight replicates of each gradient were performed, and the gradient that 100% of the target band was amplified in both experiments was used as the sensitivity gradient.

2. Reagent preparation

Taking n × 10 μ L of fluorescent PCR reaction solution, n × 1 μ L of primers, and n × 4 μ L of sterile H₂O (n is the number of reaction tubes) are mixed. Shaking and mixing for several seconds, and then instantaneously centrifuging.

And (3) placing 15 mu L of the mixed solution into a PCR tube, then respectively adding 5 mu L of the diluted plasmid standard substance into the PCR reaction tube, covering the tube cap, and immediately carrying out PCR amplification reaction.

3. PCR amplification reaction

The recommended PCR amplification procedure is: 5min at 95 ℃; reacting at 95 ℃ for 10s, at 60 ℃ for 20s and at 72 ℃ for 10s for 45 cycles; 5s at 95 ℃; 1min at 65 ℃; 65-97 ℃ (heating rate is 0.11 ℃/s); 30s at 40 ℃.

4. The result of the detection

The sensitivity standard of the method of the kit is less than or equal to 5 multiplied by 10⁴copy/fluorescent PCR reactions. The sensitivity of 35 pairs of primers in the kit is 5-5 multiplied by 10 by using a plasmid standard substance²copy/fluorescent PCR reactions (as shown in table 5) achieved the primer sensitivity criteria. Therefore, the mixture of 35 pairs of primer combinations in the kit has better detection sensitivity.

TABLE 5 kit sensitivity test results

EXAMPLE 4 use of the multiple arbovirus and invertebrate infectious Virus detection kit

1. Experimental methods

Sample source: insect HighFive cells and sf9 cells were from Beijing Yi Qiao Shenzhou technology, Inc.

Sample treatment: the detection samples are HighFive cells and sf9 cells, and the detection cell amount is about 5 multiplied by 10⁶And (4) cells. Before extraction, the cell samples were centrifuged (1000rpm, 5min), the supernatant was removed, and the pellet was resuspended in 140. mu.L PBS and extracted for further use. The Kit was extracted with the QIAamp Viral RNA mini Kit (Qiagen, cat. No.52904) and Viral nucleic acid extraction was performed according to the Kit instructions.

Depending on the type of virus, the fluorescent PCR assay is performed directly for DNA viruses, or for RNA viruses after reverse transcription of viral RNA into cDNA.

Preparing a reagent: taking n × 10 μ L of fluorescent PCR reaction solution, n × 1 μ L of primers, and n × 4 μ L of sterile H₂O (n is the number of reaction tubes) are mixed. Shaking and mixing for several seconds, and then instantaneously centrifuging.

Sample adding: and (3) putting 15 mu L of the mixed solution into a PCR tube, then respectively adding 5 mu L of nucleic acid into the PCR reaction tube, covering the tube cover, and immediately carrying out PCR amplification reaction.

2. PCR amplification reaction

The recommended PCR amplification procedure is: 5min at 95 ℃; reacting at 95 ℃ for 10s, at 60 ℃ for 20s and at 72 ℃ for 10s for 45 cycles; 5s at 95 ℃; 1min at 65 ℃; 65-97 ℃ (heating rate is 0.11 ℃/s); 30s at 40 ℃. Quality control: each control of the kit is required to meet the following requirements, otherwise the experiment is deemed invalid.

Control CT value

H2O N/A, 40 or a melting temperature of unspecific value

Positive control substance not more than 35

TABLE 6 results of the experiment

Sample(s)	CT value	The result of the detection
			Highfive cells	N/A	The detection results are all negative
sf9 cell	N/A	The detection results are all negative

As can be seen from the data in Table 6, the tested samples of the HighFive cells and the sf9 cells detected in the examples have good quality and are free from infection of arbovirus and invertebrate virus. The safety of the insect cell production system monitored by using the kit has the advantages of simplicity, rapidness, high detection sensitivity and reliable and stable result.

Sequence listing

<110> China food and drug testing research institute of Beijing Yiqiao Shenzhou science and technology Co

<120> a kit for detecting various arbovirus and invertebrate infectious viruses

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cgtctaaatt tcacatcata ggct 24

<210> 29

<211> 21

<212> DNA

<213> artificially synthesized-Baculoviridae-1F

<400> 29

tcaaggacat cgccaaccag t 21

<210> 30

<211> 21

<212> DNA

<213> artificially synthesized-Baculoviridae-1R

<400> 30

gccgctccag ttacacacaa g 21

<210> 31

<211> 19

<212> DNA

<213> artificially synthesized-Baculoviridae-2F

<400> 31

accaccaccg atgtccttg 19

<210> 32

<211> 20

<212> DNA

<213> artificially synthesized-Baculoviridae-2R

<400> 32

taactgcgaa tgtgcgtcta 20

<210> 33

<211> 23

<212> DNA

<213> Artificial Synthesis of Polydnaviridae-1F

<400> 33

gtggacagca actaatggaa gat 23

<210> 34

<211> 21

<212> DNA

<213> Artificial Synthesis of Polydnaviridae-1R

<400> 34

gactcaggag acgaaaccga t 21

<210> 35

<211> 24

<212> DNA

<213> Artificial Synthesis of Polydnaviridae-2F

<400> 35

tctaactaca tccaygcraa ctat 24

<210> 36

<211> 25

<212> DNA

<213> Artificial Synthesis of Polydnaviridae-2R

<400> 36

gatacttgtt acrgtgacyg taaat 25

<210> 37

<211> 20

<212> DNA

<213> Synthesis of-Parvoviridae-1F

<400> 37

gaaagcagcc agacaacagt 20

<210> 38

<211> 20

<212> DNA

<213> Synthesis of-Parvoviridate-1R

<400> 38

tctcaccatc ggaagaaacg 20

<210> 39

<211> 21

<212> DNA

<213> Synthesis of-Parvoviridae-2F

<400> 39

atccacgatt gctcttacac c 21

<210> 40

<211> 23

<212> DNA

<213> Synthesis of-Parvoviridae-2R

<400> 40

tcccactttc tcctctaacg tat 23

<210> 41

<211> 21

<212> DNA

<213> Synthesis of-Parvoviridae-3F

<400> 41

cgagccaatc agaatcagag a 21

<210> 42

<211> 21

<212> DNA

<213> Synthesis of-Parvoviridae-3R

<400> 42

gttgcgtata tgttccgtgt c 21

<210> 43

<211> 26

<212> DNA

<213> Synthesis of-Parvoviridae-4F

<400> 43

gaggactgga gatacatcgc cacaac 26

<210> 44

<211> 23

<212> DNA

<213> Synthesis of-Parvoviridae-4R

<400> 44

gcacttcctg cgcttgtcgc act 23

<210> 45

<211> 26

<212> DNA

<213> Artificial Synthesis of Birnaviridae-F

<400> 45

acaaccatac tratgaacaa rtgg 24

<210> 46

<211> 23

<212> DNA

<213> Artificial Synthesis of Birnaviridae-R

<400> 46

tcccasccca rcatgtccat 20

<210> 47

<211> 20

<212> DNA

<213> Artificial Synthesis of Reoviridae-1F

<400> 47

accttcagag caggctcatt 20

<210> 48

<211> 23

<212> DNA

<213> artificially synthesized-Reoviridae-1R

<400> 48

ttcgtagttt gataccatac atc 23

<210> 49

<211> 22

<212> DNA

<213> artificially synthesized-Reoviridae-2F

<400> 49

gctctatcgt atcctgactc tc 22

<210> 50

<211> 22

<212> DNA

<213> artificially synthesized-Reoviridae-2R

<400> 50

cctcacattc ttcttctgaa ct 22

<210> 51

<211> 22

<212> DNA

<213> Artificial Synthesis of Reoviridae-3F

<400> 51

tagttacctc accgctgacc tc 22

<210> 52

<211> 22

<212> DNA

<213> artificially synthesized-Reoviridae-3R

<400> 52

ccattgtcag caactaaacc at 22

<210> 53

<211> 20

<212> DNA

<213> Artificial Synthesis-nodal-1F

<400> 53

caacgggctg tcatygacaa 20

<210> 54

<211> 20

<212> DNA

<213> Artificial Synthesis-nodal-1R

<400> 54

aaacggcgtg cccrttrtct 20

<210> 55

<211> 20

<212> DNA

<213> Artificial Synthesis-nodal-2F

<400> 55

caagtcgcag atccacaaag 20

<210> 56

<211> 20

<212> DNA

<213> Artificial Synthesis-nodaviridae-2R

<400> 56

cgtcggaatg aaagtgaagc 20

<210> 57

<211> 21

<212> DNA

<213> Artificial Synthesis-nodaviridae-3F

<400> 57

caggtaagac cccaaagcaa g 21

<210> 58

<211> 20

<212> DNA

<213> Artificial Synthesis-nodaviridae-3R

<400> 58

atgtatgcgg ctttgaccac 20

<210> 59

<211> 30

<212> DNA

<213> Artificial Synthesis of-Picornavirales-1F

<400> 59

gtttaattct ttacaagayk cttttaattc 30

<210> 60

<211> 26

<212> DNA

<213> Artificial Synthesis of-Picornavirales-1R

<400> 60

acagcaggtg taatrtgkga ratatt 26

<210> 61

<211> 20

<212> DNA

<213> Artificial Synthesis of-Picornavirales-2F

<400> 61

gcattcatcg actttcccac 20

<210> 62

<211> 20

<212> DNA

<213> artificially synthesized-Picornavirales-2R

<400> 62

taaacaggcg gagcactacc 20

<210> 63

<211> 28

<212> DNA

<213> Artificial Synthesis of-Picornavirales-3F

<400> 63

atggatgata cgcacagtat tattcagt 28

<210> 64

<211> 20

<212> DNA

<213> Artificial Synthesis of-Picornavirales-3R

<400> 64

ttttgtgcyt twccdccagc 20

<210> 65

<211> 23

<212> DNA

<213> Artificial Synthesis of Tetraviridae-1F

<400> 65

actggaggca ctaacaatac aag 23

<210> 66

<211> 21

<212> DNA

<213> Artificial Synthesis of Tetraviridae-1R

<400> 66

atactgacgc cagacccttt c 21

<210> 67

<211> 23

<212> DNA

<213> Artificial Synthesis of Tetraviridae-2F

<400> 67

gcgaacgtgg ataayaagga rat 23

<210> 68

<211> 20

<212> DNA

<213> Artificial Synthesis of Tetraviridae-2R

<400> 68

ggcatgttag cytcacawgt 20

<210> 69

<211> 22

<212> DNA

<213> Artificial Synthesis of Tetraviridae-3F

<400> 69

ggacggagaa tacaagacga gc 22

<210> 70

<211> 20

<212> DNA

<213> Artificial Synthesis of Tetraviridae-3R

<400> 70

tgcctgaaga acgggagatg 20

<210> 71

<211> 266

<212> DNA

<213> artificially synthesized-PGEM-Reoviridae (orbiviruses)

<400> 71

atgtcagctg cgatacttct tgcacccggt gacgtgatca agcgttcatc cgaggagtta 60

aaacagaggc agattcaaat taatttggtt gattggatgg aaagcgagag cgaaaaagag 120

gataaaacag gatccaaaga ggaggataaa gctgaggagt caaaagatgg tgaggggacg 180

caatcggaga gcggccagaa gaaagagagc ggcaaagagg ctaaagatgc agatgtggat 240

agacgcatac atactacagt gggatc 266

<210> 72

<211> 265

<212> DNA

<213> artificially synthesized-PGEM-togaviruses

<400> 72

acatgaaatc agacacgctg cagtacacca gcgacaaacc accgggcttc tacaactggc 60

accacggcgc agtccagtat gagaatggga gatttaccgt accgagagga gtgggcggga 120

aaggcgacag cggaagaccg atcctggaca acagaggcag agttgtggct attgttctag 180

gaggtgcaaa tgagggcacg cgtacggcgc tttcagtggt cacttggaac cagaaagggg 240

tgaccattag ggataccccc gaagg 265

<210> 73

<211> 290

<212> DNA

<213> artificially synthesized-PGEM-flaviviruses

<400> 73

cacctgcatc tacaacatga tgggaaagag agagaagaag cctggagagt tcggcaaagc 60

taaaggcagc agagccatct ggttcatgtg gctgggggcc cgcttcctgg agtttgaagc 120

tctcggattc ctcaatgaag accactggct gggtaggaag aactcaggag gaggagttga 180

aggcttagga ctgcagaagc tcgggtacat cttgaaggaa gttggaacaa agcctggagg 240

aaaggtttac gctgatgata ccgcaggctg ggacacacgc atcaccaaag 290

<210> 74

<211> 276

<212> DNA

<213> Artificial Synthesis of-PGEM-Bunyavirus-1

<400> 74

cacatgtcat gtgttaggtt cttccacaat accatattac ctggcaatat gataacctca 60

atgtgccaga atatagaatt aataattatc ttagggctaa cattagccat attcatatta 120

atggttatgc ttacaaaaac ttatatatgt tatttactga tgccaatttt catgccgata 180

gcttatcttt atggttgggc ctacaataag agctgcaaaa agtgcaactg ttgtggttta 240

gcataccatc catttactaa ttgtgggtca cattgt 276

<210> 75

<211> 277

<212> DNA

<213> Artificial Synthesis of-PGEM-Bunyavirus-2

<400> 75

agagtgtgat gtcggatttg gtgttttatg atgtcgcatc aacaggtgca aatggatttg 60

atcctgatgc agggtatatg gacttctgtg ttaaaaatgc agaattactc aaccttgctg 120

cagttaggat cttcttcctc aatgccgcaa aggccaaggc tgctctctcg cgtaagccag 180

agaggaaggc taaccctaaa tttggagagt ggcaggtgga ggttatcaat aatcattttc 240

ctggaaacag gaacaaccca atcacgcatc accaaag 277

<210> 76

<211> 258

<212> DNA

<213> artificially synthesized-PGEM-asfaviruses

<400> 76

cggcatgctt gagcactaca tgatcgccaa agtcatctat gccatgatgg gcaacaagtt 60

tgtggtggac gtggactcaa acgggaagta cgtttggttc gaatttgtgc taccgggcca 120

gccaatgaat cagggagaaa tatggaagtg gcgcaaagag gtaaacccgg atgagctgca 180

catctatatt tccgaaaact tttcaagggt gatggaccga atcacggagc acatcaaata 240

ccacctcagt cagcccca 258

<210> 77

<211> 1380

<212> DNA

<213> Artificial Synthesis of-PGEM-Ascoviridae

<400> 77

gggaattcca atgacttcaa acccagaaac gccagtggta tctaatgtga ctctaagcga 60

gctacgttct gacgtgaaca agcgaggtac tatagatagt tatatctacg gaccggacga 120

gcaggtgacg acttatttcg ttcgagagat aaggccttgt gccgcgttca gcaaaatgcc 180

ggtccttttg aacaccggga atggcagtaa caagtttggc ggtacgttta cgatgcccat 240

aaacgcaagt ggagactatc tactagctct aactgcttac atctcaatca gtgccggaaa 300

gcttcgcagt tacgctgccg gtacggaatc atcatatttc ccgaaacttg ctcacaagtt 360

aatcaaatcc gtacgcctga aggttgatgc taaaccattg gttgagctga gctcgaattt 420

tatggacacg tggtccgagt tcatgatcga cggcggcaat tacgaagctt atacgaatat 480

ggtaggaggt gatttccaat acagccgttc cgttgacatt ggtgcgaaaa cactcgttct 540

accgattcca ttatatttca gtcgcgacag tggagtggcc ctacccatag gtatgatggt 600

caacaatcgc gtaacggtgg agtttgtatt tagaaaacta agcgatttgt tgatcaaaga 660

aaatgtaccg gatacgggcg ctggaacagg tttcatgaac atgctgacgg atggagattt 720

cgcaacacct cctgagatta caaagttcga ggtggtggcg gactttgcag ttgttaccga 780

ctttgaaatc gacagaacct catgctcacc acactacatt ctcatggaga agccaacgga 840

catagcggct actgaaatac tgaaaccgac cgaagcagga agcacactct cgtacgacat 900

tgagcactcg agtggtattt tgaaggcttt gtttttcggc gttaggaaca tcacgcactc 960

ggagtatctc gactactacg aagtgggcct accgaaaagc aacaacggtg tcagatcaat 1020

cggtgtttca gcactatcga aaatcggtat caagtgtggc gaccggtaca gggtcccgat 1080

gcttccggcg gagcactttg tctttatgga gccctatcgt gcggcgtgtc gagtacccac 1140

acggaaccgt gggcagtaca tgtactcttt cgcgctggat ctcaaaacgg ttgacccgaa 1200

aggttccata aatccatcga actttaacag tagcatatca gttgtaatcg aacctagcga 1260

ggcgttgagg aattcagcgg agaccttcga gtttacagct atagcactca cgagctacat 1320

tttgtacatc gacaacggta gtctaaagaa gatcgacaac ggaggcgatt tcaatgagga 1380

<210> 78

<211> 1404

<212> DNA

<213> artificially synthesized-PGEM-Iridoviridae-1

<400> 78

atgtctattt cttcgtcaaa tgtaacttct ggtttcatcg atatcgccac taaagatgaa 60

attgaaaaat atatgtatgg tggtaaaaca tccacagctt atttcgtaag agaaactaga 120

aaagcaacat ggttcaccca agtacctgtt agtttaacta gagcaaacgg atcagctaac 180

tttggaagcg aatggtctgc atccatttct agggccggag attatttgtt atatacttgg 240

ctaagagtga gaattccatc tgtaactttg ttatcaacaa atcaatttgg tgccaatggt 300

agaattaggt ggtgtagaaa ttttatgcat aacttaatca gagaatgtag tattacattt 360

aacgatttgg tagcagcaag atttgatcat tatcatctag atttttgggc cgcatttaca 420

acacctgcgt caaaggcagt tggttacgat aatatgattg gaaatgtttc tgctcttatt 480

caaccacaac ctgtacctgt tgctcctgca acagtaagtc ttcctgaagc tgacttgaat 540

ttgccattac ctttcttctt ttcacgcgat tctggagttg ctcttcctac tgctgctttg 600

ccatacaatg aaatgagaat taatttccaa ttccatgact ggcaacgctt gttgattctt 660

gacaacattg cagccgttgc atcacaaacc gttgtacctg ttgtaggtgc tacaagtgat 720

attgcgacgg ctcctgttct tcatcatgga actgtatggg gtaactatgc tattgtctct 780

aatgaagagc gaagacgtat gggatgctct gtaagagata ttttggttga acaagttcag 840

actgctccac gtcatgtttg gaatccaaca actaatgatg ctcctaacta cgatattcgc 900

ttctctcatg ctattaaagc acttttcttt gctgttagaa atacaacctt ttctaatcaa 960

ccttcaaact atacaaccgc ttctcctgtt attacgtcaa caactgttat tttagagccc 1020

tcaaccggtg cttttgatcc tatacatcat actaccctta tttatgaaaa tacaaatcgg 1080

ttgaatcata tgggcagtga ttacttttcg ttggttaatc catggtatca tgcgcctaca 1140

attcctggac ttaccgggtt ccatgaatat tcatattcgc ttgcctttaa cgaaattgat 1200

cctatgggaa gcaccaatta tggtaagctt accaatatta gtattgtacc aactgcaagc 1260

cctgctgcta aagttggtgc tgctggtact ggtcctgctg gaagtggaca aaacttccca 1320

caaacattcg agttcattgt tacagcatta aacaacaata tcataagaat ttccggcgga 1380

gcacttggtt ttcctgtcct ctag 1404

<210> 79

<211> 253

<212> DNA

<213> artificially synthesized-PGEM-Iridoviridae-2

<400> 79

tgtgggggcg tggtaccacg ggctgacgag cgagtagtaa tcgcttccca tggttcccag 60

tcggttcgag ttttcgtaaa tgagcgttgt ggtggcaatg ggatcaaacg atcccgacgg 120

ctcatagttg accgaggttc cgctaactac tggtgaactc gtaccatagt tggaccattc 180

cgcggcactt gtcttgttgc ggacggcaaa gaagagcacc ttgacggcgt gcgagaagcg 240

aatgtcaaac gac 253

<210> 80

<211> 249

<212> DNA

<213> artificially synthesized-PGEM-Iridoviridae-3

<400> 80

cgcagcttta ccttacaatg agatgcaaat taactttaac tttagagatt ggactgaact 60

acttgttctt acaaatagtg ctttagttcc tcctgcgagc ccatatgtcc caattgtggt 120

tccaactcat cttacaactg ctcctgtttt aggacctgtt caagtatggg ctaactatgc 180

tattgtaagt aatgaagaac gtcgtagaat gggttgtgct attagagaca tcctaattga 240

acaagttca 249

<210> 81

<211> 260

<212> DNA

<213> artificially synthesized-PGEM-Iridoviridae-4

<400> 81

gtatttcaga acaaagacac cggaaatgtg attcctatct ctgctaccga catagccggc 60

gggctcgccg acaccgtgga agcttacgtg tacatgaccg tgggtctcgt ttccaacgtg 120

gaaaggtgcg ccatggcagg gaccgtcagg gatatggttg tggaacaaat gcaggccgcc 180

cccacacaca tcgttaaccc tcaaaataca aataacgtcc acgtagacat gaggttctcg 240

cacgccgtga aagccctctt 260

<210> 82

<211> 1056

<212> DNA

<213> Artificial Synthesis of-PGEM-Poxviridae-1

<400> 82

atgaataaat tctattatat atgtatttat atcaatattt tatacgtctg cgttagcggg 60

catggatata tgacatttcc tatagccagg cagagaagat gttcggtaag gggaggtcag 120

tggtggccac ctaacggaga tggaataact gatactatgt gtagggccgc ttatcaaaat 180

gtatataata aagtattaaa tcaatataat gacccacaag aggctgctac tgctgctcaa 240

tatatgttcc aacaggataa tgaatatgct gccttggcag gacccgatta tactaattta 300

tgtaatttgc aacaaaatgt agtacctaac aatttatgcg ccgcgggagc cgatgattgg 360

gatgtagttc cattcggaga taagagtgga atggatttgc caggaaattg ggtacctacc 420

gttattcctc ttgatagcaa ccaccaatct agcgtagctc tagaattgga attttgtcct 480

actgctgttc atgatcctag ctattatgaa gtttatatca ctaattcggg atttaatgta 540

cataccgata atgtagtatg gggtaaccta gaattgatat ttaatgatac agttccatta 600

agacccaaat ctagtacttc gacttgtaat gctaatccaa acgtttatag atttacagta 660

tctattcccg taagacctgc tcaatttgta ttatatgtaa gatggcaaag aatcgatccc 720

gtcggtgaag gattttataa ttgtgttgac atggcattcg attatgctgc cggaccttcc 780

gaagaagatg taatatatcc agattacgag gctcctggac agaatgcata cacttgtcat 840

gctaatagaa ataaatacgg aggaaattat gaaaatacta tcgatgaaga taaatatcag 900

gctcagttag atgaatctat aaagagtaga tacgacaaat atagtagaca taaaggagga 960

aaattcggac aaaaacaatg taatggtaat aaacaccatt ataataaata taccaaatat 1020

tataaccaaa attataaaaa taacaaaaat tattaa 1056

<210> 83

<211> 265

<212> DNA

<213> Artificial Synthesis of-PGEM-Poxviridae-2

<400> 83

caccaatgta aatatactcc caaatgtcca ttccaatttg ttgtaaacag cccagatact 60

acgattcact tatatggtat ttctaatgtt tgtttaaaac ctaaagtacc caaaaattta 120

agactttggg gatggatttt agattgcgat acttctagat ttattaaaca tatggctgat 180

ggatctgatg atttagatct tgacgttagg cttaatagaa atgatatatg tttaaaacaa 240

gccataaaac aacattatac taatg 265

<210> 84

<211> 270

<212> DNA

<213> Artificial Synthesis of-PGEM-Poxviridae-3

<400> 84

cgtatgcagt ttgtgcgggt tttcacttat atgatcttcg agattactcg ttaaactttt 60

tacggaaaag atgattttct gacaacgaaa agtcggaccc acgctgattg cgtatttatc 120

ccttaccaag aactatcttt tcggttggat cctttgctgc cggcttttaa aatctttaag 180

ataccgaaac aatcttcaat ttcaaattcc atatcattga agaaattaaa gcctatgatg 240

tgaaatttag acgtgattga ctactacaac 270

<210> 85

<211> 266

<212> DNA

<213> Artificial Synthesis of-PGEM-Polydnaviridae-1

<400> 85

agcccagctg catcgataat tggaaatatt gtcggggtat caacaagccc tgctgtggac 60

agcaactaat ggaagatggt acgctcggac cgaagcactt cgtttgtttt gaactcggac 120

aggggatctg tacaccactt tctcagatta aaaatgcaga cttatttgct aagttggccg 180

accaattaaa cgacactaat tatgccgaat tggaatccca atattggaaa tcggtttcgt 240

ctcctgagtc tagttctact ttctaa 266

<210> 86

<211> 266

<212> DNA

<213> Artificial Synthesis of-PGEM-Polydnaviridae-2

<400> 86

tcgaattttg ggatactact agagtgtgtt taaaacagaa gcgatctaac tacatccatg 60

cgaactatct tgatggtttt gaacgagtcc ggaaattcat agtgacccaa gaaccgatgg 120

acaacacgct cgagaactac tggagaatga tttggcagac tggcactagg gttattgtga 180

tgttgaatgg tgccgacgaa cctgcagtgc cctctgtaag tgactattcc gaattgaaag 240

gatttacagt caccgtaaaa agtatc 266

<210> 87

<211> 266

<212> DNA

<213> artificially synthesized-PGEM-Baculoviridae-1

<400> 87

tactgggtga tgtgaggcac aaggaggaat tgattcgcga agcacaattc gacccaatca 60

aggacatcgc caaccagtac atggtgactg aagatccctt cagaggccct ggtaagaatg 120

ttaagattac cctgtttaag gagattcgtc ggatccagcc agataccatg aagcttgtgt 180

gtaactggag cggcaaagag tttttgcgcg aaacctggac tcgtttcatc tcagaagagt 240

tccccatcac caccgatcag gaaatt 266

<210> 88

<211> 1242

<212> DNA

<213> artificially synthesized-PGEM-Baculoviridae-2

<400> 88

atgacgcacg tcaccgtgaa agaatactta cagagtctta attatcaacc gcatccaatg 60

gattttgtgc gcagactcaa tttcagtaat gctgttatcg gcgacagaat cgtcatggac 120

agagagcttg ctataaaata tttacgtctg gcagaatcta tttatcaaaa tacagttaaa 180

atagccatcg acgtcccggc caacgtcccg gccgttttac ccgataatgt gcgcataaag 240

aataaaattt tgcaactcga aaatatggtg acccgcgtcg acgccaatag ccgttataaa 300

actattttat tcgacataat cgataaaatt aaccacgaga atgaactctc aaaaatagaa 360

aatctcatga aaaccttttt caaactttat aaacagtatc aagatgaagt cgaaaaaccg 420

acagctaaaa tcgattcgat tttcaatcaa attataaatc tagatacgtc ggcggctacg 480

ccatttactc cgacgtcggt gtcaccccca tcgatttccg taccaatcac cgagacacca 540

gaagcaaaaa taaatttcat cccggaaaaa aatataattg taccatcgtc gccgacgcaa 600

acacaaatta cgataccagc gtcaccacct ccgccgccaa tgtcgacgac ttcgatgcca 660

ccgccgccac caccgccgcc accaccacct ccaccaccac cgatgtcctt gataccgcct 720

ccgccaccac ctccgcccat gtttcaaatg tttaaaaatg acgaaaataa agaaatttcg 780

tcagcaaaaa catcgtctaa acctacgaca attgcggcgg cgccgataga cgcacattcg 840

cagttaatgg aagcaattcg atctggcacc attttaaaaa atgtaaaaaa aattaccgca 900

gagaaagaac cgattgttga gacagcgcct tccatattac atttggctct agccaaaaga 960

gtaaaagctt tagccgaaag cacggataat gaggaatctt cgttttcaga ggacaataac 1020

gaatggatca gcgacaatga gctcgaaacc atcaaaaaca agtatttaaa cactatgaat 1080

agagttcaga acagtggcat tgaaaatgat gaaattttac gcatgtcctc taatgtgtcg 1140

agattgataa atagtcaata ctttagcaaa caggatatta aacaagccga gagactttta 1200

acggaaataa acgaaaaagt cgataatgaa tctacagctt ag 1242

<210> 89

<211> 1617

<212> DNA

<213> Synthesis of-PGEM-Parvoviridae-1

<400> 89

atgcaagatg gaaagcagcc agacaacagt aactatgtcc cagctttgga tgacactatg 60

ggaacggtat ggcaacgaat gcgtgacgat gccgggtttt tggatgagac tgctagagac 120

ggatttggag gattcttctc aagaggagtg gaaaacagcg tttcttccga tggtgagata 180

ttgggcccgc agcaacaact tcaacgtgac atcgtatcaa gaattaaaag aacgcctttc 240

ggaccttttc gccggtacct tagcgacgtc ttggcctgcg acgactcaag agaagctaat 300

agaatcgcta gacaagttat tagaacagct agagattacc caggaaattt gtgccttata 360

tcaacccacg acgatcatgt ccacgtcgtc catgactgcg cgtattccga tggcagctgt 420

agatgcaaaa tcctcaaaga agagacagtt aaagctaagc gtcgcggagc gattagaaaa 480

cgtaaagcga ttagctcaat cgcatcagat cagtgggacg atatcatcct ctatttcatt 540

tccaacggac ggtggcttca gttcgctaaa gtgggagggt ccgtggtcgg attaccgtat 600

ggatatcaag ctttacagga gcgaggacat caaggacaag actccggaca aatattggga 660

acatgcactt ctacgggctc aagtgaatta tgcggagaat catccattga agaagttact 720

ggaaaaagtg caagaattaa tagtagagat ggcagaagga gaagacgacg tgcaagtgac 780

atccgcgaag aaatggagaa aatagtgaat gaaaatcccg tgtgtccatt agcaggaatt 840

atatctacaa gacaatggtt aactcataaa gacttacgat acttacgtgc agataatgaa 900

actgtgaaat cctttcttga tgctaaaagt gaggaaatgt gttattggac tttgcatgac 960

tttaatactt tttattcaca accatattgt aatccaacat ttatggctgg ttatcaacca 1020

ttagaagata aatactattc tgttaatgat tcattgacaa ttttaaataa gctattagca 1080

tatcagtttg atgatgacgt tgttcagatt aaatcatttt taaatacatt ttataatgtg 1140

cttgagcgta aattaccaaa gtgtaataca atatgtgtat ggtctcctcc aagtgcagga 1200

aagaacttct tttttgatgt gtatctacat tatctaatga actatggaca attaggtata 1260

atgaataaga ctaataattt cagtttacaa gaagcaacat ctaaacgtgt gcttttgtgg 1320

aatgaaccaa actatgagga tgcatataca gacactttga aaatgttaac aggtggtgat 1380

gctctatgtg taagagtaaa acaaaaaaaa gattgtcatg tatataagac accacttatt 1440

gtacttacaa ataatatgat tggattcatg catgaactag catttgtaga tagagtgaag 1500

gtatatagat ggaaacaagc tccattccta gctgagtata ataagaaacc taatccgctt 1560

gtagcttttg aaatattggt gtattgggaa ataattccaa ggataattga acaataa 1617

<210> 90

<211> 307

<212> DNA

<213> Synthesis of-PGEM-Parvoviridae-2

<400> 90

tgcgtgagta cggaagaagt agacgaaacg gactgttcgg attttctgaa gaaggagatc 60

acatccacgt catccacgat tgctcttaca ccaatcgcag ttgcagggac atctggatta 120

gtcaagtcaa gcccttcgga tctgttcaga aaactggcaa accagtcaaa ttcatctggg 180

aattcaagcg aacagactgg tacgatgtct tcatctattt ctttgtacga aaacggggag 240

agcgtgcaat atacgttaga ggagaaagtg ggaaaatacc gagtaacgat gaatgtgtac 300

gatggac 307

<210> 91

<211> 301

<212> DNA

<213> Synthesis of-PGEM-Parvoviridae-3

<400> 91

ccagcaatcg agccaatcag aatcagagag ggaggaggag aatcgcagga accgaccatc 60

gagttcaaga catataaata ctactcggaa aagaaagtct atcacaacat cgaagggcgt 120

gcttacgaag aaaaaaagtt tgaagaacca acccagggcc atttccacat acttcacgcc 180

tgcagatggt acaataatga atgcaggtgc ctcgggagga gctttgacgt caatccaagg 240

aagaataagg caatccctat cgacccggtc gacacggaac atatacgcaa cattatgttc 300

t 301

<210> 92

<211> 266

<212> DNA

<213> Synthesis of-PGEM-Parvoviridae-4

<400> 92

gtcgatctcg tcgacggaga gttggatgga tcaactggat cgaatcgaga aacagcatac 60

tacacattcg tcctccacaa agacaacgtt aaagaggact ggagatacat cgccacaaca 120

agggccaagc aagcgccgag tttcatcaca ttcgatcacg gagaccacat ccatatcctc 180

ttctcctcgt caaatacagg aggaaacagt accagagtca gaaccagaat caccaagttt 240

cttagtgcga caagcgcagg aagtgc 266

<210> 93

<211> 387

<212> DNA

<213> artificially synthesized-PGEM-Birnaviridae

<400> 93

ttaaagaact tccagataaa gaatcctgga cagggcagtg gcaatccgtg gaccttcttg 60

aacaatcatg tattaaccac gatactgatg aacaagtggt ctgagatagg taagccacaa 120

ccctccccgg atgttatcga gaagttggca tcaatgacag gaattgattt caaggttgaa 180

ctagtagtga caaacttccg agagaaacta atagcagctt caaggcattc gatccctaca 240

agtaacaggg ttgaacccag aacaattgta gagatggaca tgctgggctg ggacgtaact 300

cacacggagt ttggcttcac accggtactg tcaaaagaac gactattcaa atcaatagcc 360

tgcccacaac caccaagtag caccttt 387

<210> 94

<211> 266

<212> DNA

<213> artificially synthesized-PGEM-Reoviridae-1

<400> 94

agtatgatcc ttcagattat ccgttagctg aagatgaatc tttctttaag caaggtcaca 60

agtacgatta cttagtaacc ttcagagcag gctcattgac caatacatat gaaccgaaaa 120

ctaagatgta caaactacat gctgccttag ataagttaat gcacgttaag caacgtaaat 180

cacgtttcgc tgatctgtgg cgcgagttat gcgcagttat cgcttcatta gatgtatggt 240

atcaaactac gaattaccca ctgcgc 266

<210> 95

<211> 266

<212> DNA

<213> artificially synthesized-PGEM-Reoviridae-2

<400> 95

acaaaccctg aattcatgag gatgtcaaat gcatttgcca gtactagaag aggaccgttc 60

tacaatattg cttcattggt accagctcta tcgtatcctg actctcgcag tgttccacta 120

atcgtcggat ttatactcac tcaagaaaac ctaagtttgc tcagtctcta cagtatgata 180

gtaaccacga aagtttcaag cacgataatg gcattatacg aaaacaatag ttcagaagaa 240

gaatgtgagg actctatttc tgccgc 266

<210> 96

<211> 266

<212> DNA

<213> artificially synthesized-PGEM-Reoviridae-3

<400> 96

taggcaagtt atcaaaagac cctttgaccc cgatagttac ctcaccgctg acctccatct 60

ttatctcaag aacgggaaaa cgttgacgat ccacatcgag cggaacctct tctttagcca 120

tgagttcact tgggaagaaa tttgtcgtgg cgcttaccgc gaacattatt tatctaactc 180

tgggcctggt aaatgcgata ctcaagaata tattgatggt ttagttgctg acaatggtgg 240

ccgttccact cacaattcat cctacc 266

<210> 97

<211> 287

<212> DNA

<213> Artificial Synthesis of-PGEM-nodal-1

<400> 97

atgactctaa aagttattct tggagaacac cagatcaccc gaactgaatt gttagtcggg 60

attgcaaccg tatctgggtg cggtgccgta gtgtactgca tatccaagtt ctggggctat 120

ggggcaattg cgccctatcc tcagagtgga gggaaccgag ttacacgcgc attgcaacgg 180

gctgtcattg acaaaacgaa gaccccgata gagacacgtt tctatccgct tgacagcctg 240

cgtaccgtga cgcctaagcg tgtcgcagac aacgggcacg ccgtttc 287

<210> 98

<211> 266

<212> DNA

<213> Artificial Synthesis of-PGEM-nodal-2

<400> 98

tggatcttgg tcccatgaag tttgggactg gtgcgcgttt ggtgagttca tcgagacccg 60

agacgcgagc tggcttgctt ggttcgcccg ggcggttgga ctcaccaagt cgcagatcca 120

caaagttcac tactgccgtc catggccgca atcgccccat cgcgctttgg tgtggtgtct 180

gcctgtagca agctactggc gcttcacttt cattccgacg gacctgcata cgcgcacgct 240

tcggcgtgtg cgttatcagg acacgt 266

<210> 99

<211> 266

<212> DNA

<213> Artificial Synthesis of-PGEM-nodal-3

<400> 99

gaagatcttg tacgagcagc cgtggtacgg cccaggtaag accccaaagc aagtaggacg 60

caggcttcag agtatcgcca aacacggtac actggagagt gactactcac gctttgatgg 120

ctcaatcagc gaatggctcc agaaaaacgt ggtcaaagcc gcatacatgc ggttcttcaa 180

agagcaccaa cgaacggagt tccagagctg gtttagcaaa gtcttcatgc agatgggcac 240

cacgactgct ggtgtgaggt acgaag 266

<210> 100

<211> 288

<212> DNA

<213> artificially synthesized-PGEM-Picornavirales-1

<400> 100

agtatgcgta ttaagatggt agccgagacg caggacggaa caggaactcc ccggaagaaa 60

acaaacttta cttggtttgt gcgtatgttt aattctttac aagactcttt taattccctt 120

atttcaacga gttcttctgc agtgacgacc acagttttac catccggcac tattaatatg 180

ggtccatcca cacaagtaat tgacccaact gtagaaggct tgatagaggt agaagttccc 240

tattacaata tttcacatat tacacctgct gtgaccattg acgatgga 288

<210> 101

<211> 266

<212> DNA

<213> artificially synthesized-PGEM-Picornavirales-2

<400> 101

tcattccaac atctctatta aaggttcctt ggcgttctcc tcaagcaggg cattcatcga 60

ctttcccact ttttcgagca ttctcctttc aacctctctc atacaatcat atttgcaacc 120

acatctccac ttctctaagt tctttacctt atccaaacta tcatagatat cttcagcggc 180

cgtcaggtag tgctccgcct gtttagagat attaattgcc ctcagctgct tcttttgaaa 240

cattgcttgc actccgagtg ctccct 266

<210> 102

<211> 266

<212> DNA

<213> artificially synthesized-PGEM-Picornavirales-3

<400> 102

ctccaaatag gatcaatacc cccatggctc aagacacttc atcggctcgg agcatggatg 60

atacgcacag tattattcag tttttacaac gccccgtact cattgaccac attgaggtca 120

ttgctggatc aacagcagat gataacaaac ccctcaatag atatgtgtta aatcgacaga 180

atccacaacc atttgttaaa tcttggacat tgccttcagt agttttaagt gctggaggta 240

agggacaaaa attagctaat tttaaa 266

<210> 103

<211> 288

<212> DNA

<213> Artificial Synthesis of-PGEM-Tetraviridae-1

<400> 103

agtatgcgta ttaagatggt agccgagacg caggacggaa caggaactcc ccggaagaaa 60

acaaacttta cttggtttgt gcgtatgttt aattctttac aagactcttt taattccctt 120

atttcaacga gttcttctgc agtgacgacc acagttttac catccggcac tattaatatg 180

ggtccatcca cacaagtaat tgacccaact gtagaaggct tgatagaggt agaagttccc 240

tattacaata tttcacatat tacacctgct gtgaccattg acgatgga 288

<210> 104

<211> 259

<212> DNA

<213> Artificial Synthesis of-PGEM-Tetraviridae-2

<400> 104

atggcagcga catcagaggc atggattcac gactatctag acccggacgg agaatacaag 60

acgagcctgg acgacgggaa aattcccgac ggcgcgatac ctcagtcaac atgcggtcaa 120

tttcgaggga ccgtgggcgc cagatacccg ggactgaatt ctacgacgct accgctggat 180

ggcgggacct ggcctctact agtgatgcat ctcccgttct tcaggcatcc gttgttgttc 240

atcaccacca ccagcaaca 259

<210> 105

<211> 316

<212> DNA

<213> Artificial Synthesis of-PGEM-Tetraviridae-3

<400> 105

cttgtctatc atctcctacc ctatgtttcg cactgcttac tttgctgtcg cgaacgtgga 60

taataaggag atatcccttg acgtcacgaa cgatcttatc gtttggctta acaatctcgc 120

tagttggcgt gacgttgttg atagcggaca gtggttcact ttttcggacg acccgacttg 180

gttcgtccgc attcgtgtgc tacaccccac atacgatctt cctgatccga ccgagggctt 240

acttcgtacg tgctccgact accgacttac ttataagtcg ataacttgtg aggctaacat 300

gcccacgctc gttgac 316

Claims

1. A specific primer pair combination for detecting a plurality of arboviruses and invertebrate infectious viruses is characterized by comprising the following primer pairs:

(1) the nucleotide sequence of the primer pair for detecting the bluetongue virus is shown as SEQ ID NO 1-2;

(2) the nucleotide sequence of the primer pair for detecting the togaviridae is shown as SEQ ID NO 3-4;

(3) the nucleotide sequence of the primer pair for detecting the flaviviridae family is shown as SEQ ID NO 5-6;

(4) the nucleotide sequence of the primer pair for detecting the bunyaviridae is shown as SEQ ID NO 7-10;

(5) the nucleotide sequence of the primer pair for detecting the hog cholera virus family is shown as SEQ ID NO. 11-12;

(6) the nucleotide sequence of the primer pair for detecting the vesicoviridae is shown as SEQ ID NO 13-14;

(7) the nucleotide sequence of the primer pair for detecting iridoviridae is shown in SEQ ID NO. 15-22;

(8) the nucleotide sequences of the primer pairs for detecting the poxviridae are shown as SEQ ID NO 23-28;

(9) the nucleotide sequence of the primer pair for detecting the baculovirus family is shown as SEQ ID NO. 29-32;

(10) the nucleotide sequence of the primer pair for detecting the multi-component DNA virus family is shown as SEQ ID NO. 33-36;

(11) the nucleotide sequence of the primer pair for detecting parvoviridae is shown as SEQ ID NO 37-44;

(12) the nucleotide sequence of the primer pair for detecting the binuclear glycogenoviridae is shown as SEQ ID NO: 45-46;

(13) the nucleotide sequence of the primer pair for detecting reoviridae is shown as SEQ ID NO 47-52;

(14) the nucleotide sequence of the primer pair for detecting the Rodaviridae is shown as SEQ ID NO: 53-58;

(15) the nucleotide sequence of the primer pair for detecting the picornaviridae family is shown as SEQ ID NO 59-64;

(16) the nucleotide sequence of the primer pair for detecting the four virus families is shown as SEQ ID NO: 65-70.

2. Use of the specific primer pair combination of claim 1 for the preparation of a kit for the detection of a plurality of arbovirus and invertebrate infectious viruses.

3. A detection kit comprising the specific primer set combination according to claim 1.

4. The kit of claim 3, wherein each detection primer detects a viral species and a nucleic acid type as follows:

(1) the virus nucleic acid type of the bluetongue virus is dsRNA;

(2) the viral nucleic acid type of togaviridae is ssRNA (+);

(3) the type of viral nucleic acid of the flaviviridae family is ssRNA (-);

(4) the type of viral nucleic acid of the bunyaviridae family is ssRNA (-);

(5) the type of viral nucleic acid of the hog cholera virus family is dsDNA;

(6) the virus nucleic acid type of the vesiculoviridae family is dsDNA;

(7) the virus nucleic acid type of iridoviridae is dsDNA;

(8) the viral nucleic acid type of poxviridae is dsDNA;

(9) the viral nucleic acid type of baculoviridae is dsDNA;

(10) the viral nucleic acid type of the multicomponent DNA virus family is dsDNA;

(11) the virus nucleic acid type of parvoviridae is ssDNA;

(12) the type of viral nucleic acid of the family of the binuclear glycoviridae is dsRNA;

(13) the type of viral nucleic acid of reoviridae is dsRNA;

(14) the type of viral nucleic acid of the nodaviridae family is ssRNA (+);

(15) the type of viral nucleic acid of picornaviridae is ssRNA (+);

(16) the viral nucleic acid type of the four virus families is ssRNA (+).

5. The kit of claim 3, comprising 35 positive control plasmid standards, each of which is:

(1) the nucleotide sequence of the positive plasmid corresponding to the primer for detecting the bluetongue virus is shown as SEQ ID NO: 71;

6. A kit as claimed in any one of claims 3 to 5, wherein the operating program comprises the steps of:

(1) extracting nucleic acid of a sample to be detected;

(2) performing a fluorescence PCR reaction on the sample nucleic acids with the specific primer pair combination according to claim 1; the result is determined from the amplification curve and the melting curve.

7. The kit of claim 6, wherein the fluorescence-PCR reaction procedure of step (2) is as follows: 5min at 95 ℃; reacting at 95 ℃ for 10s, at 60 ℃ for 20s and at 72 ℃ for 10s for 45 cycles; 5s at 95 ℃; 1min at 65 ℃; the temperature rise rate is 0.11 ℃/s at 65-97 ℃; 30s at 40 ℃.

8. The kit of claim 6, wherein the fluorescence-PCR reaction of step (2) is performed in a 20 μ L reaction system comprising: 10 mu L of fluorescence-PCR reaction solution, 1 mu L of primer pair working solution, 5 mu L of sample to be tested and 4 mu L of sterile H2O 4.

9. Use of a specific primer pair combination according to claim 1 or a kit according to any one of claims 3 to 8 for monitoring the safety of insect cell production.

10. Use of a specific primer pair combination according to claim 1 or a kit according to any one of claims 3 to 8 for controlling the safety of a pharmaceutical product in the field of pharmaceutical product manufacture.