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isknv gene intron and its application in distinguishing isknv live virus and inactivated virus

An intron, live virus technology, applied in the direction of double-stranded DNA virus, virus, virus/phage, etc., to achieve the effect of good specificity, high sensitivity, and shortening the period of inactivation test

Active Publication Date: 2020-09-15
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the genome of ISKNV has been published (NC_003494.1). The genome is 111362bp in length and contains 124 ORFs, but no articles have reported that it contains introns
Therefore, there is no relevant report on the method and kit for distinguishing ISKNV live virus from inactivated virus by RT-PCR method targeting the viral gene of the intron

Method used

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  • isknv gene intron and its application in distinguishing isknv live virus and inactivated virus
  • isknv gene intron and its application in distinguishing isknv live virus and inactivated virus
  • isknv gene intron and its application in distinguishing isknv live virus and inactivated virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Screening and verification of intron IN-3

[0052] 1. Screening and primer design of intron IN-3

[0053] According to the results of transcriptional profiling of CPB cells infected with ISKNV in our laboratory, compared with the genome sequence of ISKNV (NC_003494.1), we screened out Unigene0020179 (ORF003L) at the position of 668bp~748bp, containing an 80bp inclusion Sub, named intron IN-3, see Table 1.

[0054] Table 1 Genome sequence information where introns are located

[0055]

[0056] Primer 5.0 software was used to design specific primers, and the primer information and expected DNA fragment and cDNA fragment sizes are shown in Table 2.

[0057] Table 2 List of PCR primers designed across introns

[0058]

[0059] 2. Intron verification

[0060] 1. RNA extraction and DNA positive template preparation

[0061] Dilute ISKNV virus stock solution 10×, 100×, inoculate CPB cells and incubate for 72 hours, then extract total RNA, each T25cm 2 The...

Embodiment 2

[0070] Embodiment 2 Nested PCR amplification

[0071] According to ISKNV, the ORF003L gene contains an intron IN-3 located at the position of 668bp-748bp and the size is 80bp. Such as figure 2 As shown, after the DNA is transcribed to generate mature mRNA, the intron will be cleaved, and nested primers (Table 3) are designed on exon a and exon b, respectively, for PCR amplification.

[0072] Table 3 is used for the IN-3 gene primer of nested PCR amplification

[0073]

[0074] The 25 μl reaction system used for the first round of amplification is: 12.5 μL 2×Taq PCR StarMix, 1 μL each of primers F3 and R3 at a concentration of 10 μM, 1 μL DNA or cDNA template, 9.5 μL ddH 2 O; Reaction conditions: 94°C for 5min, 94°C for 45s, 68°C for 45s, 72°C for 90s, 30 cycles, 72°C for 10min, the product was detected by 2% agarose electrophoresis.

[0075] The 25 μl reaction system used for the second round of amplification is: 12.5 μL 2×Taq PCR StarMix, 0.5 μL each of primers F3’ and...

Embodiment 3

[0077] Embodiment 3 nested PCR method sensitivity test

[0078] Dilute the ISKNV gradient to 10 0 、10 1 、10 2 、10 3 copy / mL, take 1mL of virus liquid to inoculate T25 cell culture flask, collect the cells at 7 days, 9 days and 11 days after inoculation respectively, prepare cDNA template according to the above method, and use the established Nest RT-PCR method for detection to determine the Methods To detect the sensitivity and minimum detection period of ISKNV mRNA, and set the cells not inoculated with virus as negative control. see results Figure 4 .

[0079] Figure 4 In M1.DNAMarker DL2000, the primers used are F3 and R3, 1. DNA positive template, 2. cDNA positive template, 3. Negative control, 4. Cell seeding 10 0 ~10 3 The cDNA samples obtained by incubation for 7 days with virus copy number;

[0080] The primers used by M2.DNAMarker DL2000 are F3, R3, 1. DNA positive template, 2. cDNA positive template, 3. Negative control, 4. Cell seeding 10 0 ~10 3 The cD...

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Abstract

The invention discloses an infectious spleen and kidney necrosis virus (ISKNV) gene intron and its use in distinguishing of live and inactivated ISKNVs and belongs to the field of virus detection. The intron is located on ORF003L of an ISKNV genome NC_003494.1, has length of 80bp and is named as IN-3. The invention discloses the intron of ISKNV. The intron is named as IN-3. Through the characteristics of transcription shearing of the intron IN-3, the intron IN-3 is used as a virus complete inactivation index, and an ISKNV inactivation rapid detection nested RT-PCR method is established. The ISKNV gene intron can shorten an inactivation test cycle in ISKNV cell inactivated vaccine production process and improve vaccine production efficiency.

Description

technical field [0001] The invention belongs to the field of virus detection, in particular to ISKNV ORF003L gene intron and its application in distinguishing ISKNV live virus and inactivated virus. Background technique [0002] Mandarin fish (Sinipercachuatsi) is an important species of high-quality freshwater fish consumption and export earnings in my country. It is deeply loved by consumers because of its delicious taste and high protein content. However, the serious disease problem has become the main bottleneck restricting the development of mandarin fish aquaculture. Since 1997, the outbreak of infectious diseases of mandarin mandarin fish has brought huge economic losses to the mandarin fish aquaculture. Wu Shuqin and others confirmed for the first time that a large spherical virus particle with a hexagonal cross section and a diameter of about 150nm is the main pathogen of the fulminant infectious disease of mandarin fish. Because the virus mainly infects the spleen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11C12Q1/70C12Q1/6848
CPCC12N2710/00022C12Q1/6848C12Q1/701C12Q2549/119
Inventor 付小哲李宁求张醴溪林强梁红茹刘礼辉黄志斌
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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