Real-time fluorescence quantitative PCR (Polymerase Chain Reaction) detection method and kit for recombinant rhabdovirus

A technology of recombinant baculovirus and primer pair, which is applied in the field of detection, can solve the problems of not being able to distinguish between empty virus particles and recombinant virus particles in the Bac-to-Bac baculovirus expression system, and difficulty in obtaining primers, etc.

Inactive Publication Date: 2013-10-30
QINGHONG BIOTECH SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is often difficult to obtain primers with excellent specificity and generality in practical applications
[0012] In summary, the current baculovirus titer detection methods have their own limitations and deficiencies, and none of them can distinguish between empty virus particles and recombinant virus particles in the Bac-to-Bac baculovirus expression system

Method used

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  • Real-time fluorescence quantitative PCR (Polymerase Chain Reaction) detection method and kit for recombinant rhabdovirus
  • Real-time fluorescence quantitative PCR (Polymerase Chain Reaction) detection method and kit for recombinant rhabdovirus
  • Real-time fluorescence quantitative PCR (Polymerase Chain Reaction) detection method and kit for recombinant rhabdovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Embodiment 1. Design and test the specific PCR primers of Bac-to-Bac baculovirus expression system recombinant virus

[0102] Design of Specific PCR Primers for Recombinant Viruses in Bac-to-Bac Baculovirus Expression System

[0103] In the Bac-to-Bac baculovirus expression system, design specific PCR primers for the 2164bp fragment of the Tn7R-Tn7L region on the pFast Bac-to-Bac vector integrated in the recombinant BacMid, so as to ensure that PCR can detect specific amplification product and minimize interference from nonspecific primer-dimer signals.

[0104] qPCR was performed on the designed PCR primers to test their specificity and amplification efficiency. After systematic testing, the best primers TRTL-3F and TRTL-3R were selected as PCR primers for final detection:

[0105] Table 1

[0106]

[0107]

[0108] Test steps:

[0109] 1. Alkaline lysis method to extract baculovirus DNA:

[0110] According to the Invitrogen company's Bac-to-Bac system instr...

Embodiment 2

[0131] Extraction of Bac-to-Bac Baculovirus DNA by Alkaline Lysis and QPCR Amplification of Samples

[0132] Experiments were carried out as described in Example 1 above, using primers TRTL-3F and TRTL-3R (SEQ ID NO.: 6 and 7) to extract Bac-to- QPCR amplification of Bac baculovirus DNA and samples.

[0133] Experimental results:

[0134] QPCR amplification curve such as Figure 4 shown.

[0135] Analysis of results:

[0136] The test results of the samples are basically consistent with the plaque data (data of samples No. 1-3), and can be amplified stably (test results of 12 samples) (the samples and corresponding Ct value data are shown in the table below)

[0137] Table 4

[0138] Hole number

Embodiment 3

[0140] QPCR method confirms that the titer of Bac-to-Bac baculovirus sample is effectively amplified

[0141] Carry out experiment as described in above embodiment 1, confirm Bac-to-Bac baculovirus sample (L1 P1 virus, L1 P2 virus and L1 P3 virus by QPCR method, (virus source is the same as before, be L1 virus in virus preparation flow process Whether the titers of the first-generation, second-generation and third-generation viruses are effectively amplified.

[0142] QPCR amplification curve such as Figure 5 shown.

[0143] Analysis of results:

[0144] QPCR identification of the finally obtained L1P1-P3 virus after virus titer optimization to confirm the presence and effective amplification of the recombinant virus is a prerequisite for the smooth progress of subsequent protein expression experiments.

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Abstract

The invention provides a primer pair for real-time fluorescence quantitative PCR (Polymerase Chain Reaction), a kit containing the primer pair and a method for detecting recombinant virus particles in a Bac-to-Bac rhabdovirus expression system by using the primer pair. The primer pair can combine a Tn7R-Tn7L region on a pFast Bac-to-Bac carrier integrated in a recombinant BacMid. The real-time fluorescence quantitative PCR detection method based on the primer pair can differentiate empty virus particles from recombinant virus particles in the Bac-to-Bac rhabdovirus expression system; moreover, the method is simple, convenient and quick to operate, high in consistency and good in repeatability and has both specificity and universality.

Description

technical field [0001] The invention relates to the field of detection, more specifically, the invention relates to a real-time fluorescent quantitative PCR detection method and a kit for recombinant baculovirus. Background technique [0002] Baculovirus expression vector system (BEVS) is an expression system that uses baculovirus as a foreign gene carrier and insect cells as receptors. After the recombinant baculovirus infects insect cells, it can perform many eukaryotic post-transcriptional processes on foreign proteins, including glycosylation, phosphorylation, acylation, correct signal peptide cleavage, proteolysis, and proper folding, and The recombinant protein can also be aggregated and positioned on the same organelle as the natural protein, and it can also perform proper oligomerization assembly, so it is an ideal carrier for expressing biologically active proteins. Because the baculovirus expression system has the characteristics of safety, high efficiency, large ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor徐艳娟高磊
OwnerQINGHONG BIOTECH SHANGHAI