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Single cell real time fluorescent quantitative RT-PCR method for detecting foot-and-mouth disease virus genome RNA

A real-time fluorescent quantitative, foot-and-mouth disease virus technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of cumbersome, multiple operation steps, increase reaction time and error probability, etc.

Inactive Publication Date: 2009-03-18
广州誉嘉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of this technology are also obvious, that is, there are many steps to lyse cells and the operation steps from the isolation of single cells to the quantitative PCR reaction are cumbersome, which will cause the degradation of RNA and thus unrealistically reflect the copy number of the target gene in the original cell.
In addition, the two-step real-time fluorescent quantitative RT-PCR also increases the reaction time and error probability to a certain extent.
[0005] All in all, so far, single-cell fluorescent quantitative PCR technology has not been applied to detect the gene copy number of viruses in infected cells, so whether it can be used to detect the amount of virus carried in infected cells is a scientific issue of our concern

Method used

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  • Single cell real time fluorescent quantitative RT-PCR method for detecting foot-and-mouth disease virus genome RNA
  • Single cell real time fluorescent quantitative RT-PCR method for detecting foot-and-mouth disease virus genome RNA
  • Single cell real time fluorescent quantitative RT-PCR method for detecting foot-and-mouth disease virus genome RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Single-cell fluorescence quantitative RT-PCR detects the copy number of foot-and-mouth disease virus genome RNA in acutely infected cells

[0042] 1. Experimental reagents and instruments

[0043] A) Reagents

[0044] a) Trypsin, MEM medium, fetal bovine serum (all purchased from Invitrogen Company)

[0045] b) Cell stock solution: Each tube contains 5-10 μl 0.9% NaCl, which is divided into PCR tubes

[0046] c) Liquid nitrogen

[0047] d) Proteinase K and RNase inhibitors (purchased from Amesco and TAKARA companies respectively)

[0048] e) One-step reaction kit (including reverse transcriptase and hot-start Taq DNA polymerase and buffer), bovine serum albumin, RNase inhibitor, RNase-free water. The former two were purchased from Invitrogen, and the latter two were purchased from TAKARA.

[0049] f) RT, FP primers, probes (synthesized by Invitrogen)

[0050] g) Trizol cell lysate (purchased from Invitrogen)

[0051] B) Instrument

[0052] a) Microinje...

Embodiment 2

[0078] Example 2 Single-cell fluorescent quantitative RT-PCR detects the copy number of foot-and-mouth disease virus genome RNA in persistently infected cells

[0079] 1. Materials

[0080] A) Acquisition of BHK-21 cells persistently infected with foot-and-mouth disease virus: establishment of persistently infected cells by weak base method (rapid selection and characteristics research of cell lines persistently infected with foot-and-mouth disease virus Gu Chaojiang, Chinese Virology, Volume 18, No. 5 Issue 2003).

[0081] B) Reagents and instruments

[0082] a) Trypsin, MEM medium, fetal bovine serum (all purchased from Invitrogen Company)

[0083] b) Cell stock solution: each tube is filled with 5-10 μl 0.9% NaCl, and divided into PCR tubes

[0084] c) Liquid nitrogen

[0085] d) Proteinase K and RNase inhibitors (purchased from Amesco and TAKARA companies respectively)

[0086] e) One-step reaction kit (including reverse transcriptase and hot-start Taq DNA polymerase ...

Embodiment 3

[0110] Example 3 Application of separation and cracking single cell technology in single cell RT-PCR

[0111] 1. Reagents and instruments

[0112] a) Trypsin, MEM medium, fetal bovine serum (all purchased from Invitrogen Company)

[0113] b) Cell stock solution: each tube is filled with 5-10 μl 0.9% NaCl, and divided into PCR tubes

[0114] c) Liquid nitrogen

[0115] d) Proteinase K and RNase inhibitors (purchased from Amesco and TAKARA companies respectively)

[0116] e) RT reaction solution: M-MLV reverse transcriptase, 5× buffer, dNTP, RNase inhibitor. The former two were purchased from Promega Company, and the latter two were purchased from TAKARA Company.

[0117] f) PCR reaction solution: PremixTaq enzyme, sterile water. Purchased from TAKARA company.

[0118] g) RT, FP primers (synthesized by Invitrogen)

[0119] h) microinjector (purchased from Narashige company)

[0120] i) PCR instrument (purchased from Biometra company)

[0121] 2. Experimental method and ...

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Abstract

The invention discloses a method for detecting real-time fluorescence quantitative RT-PCR of a single cell of foot and mouth disease virus genome RNA. The method utilizes a microinjection instrument to separate single cell of a foot and mouth disease virus; and after cracking, the fluorescence quantitative RT-PCR is used to carry out quantitative analysis. Visible operation of the microinjection instrument can rapidly and accurately separate out the single cell; and the microinjection instrument is combined with the high-sensitivity fluorescence quantitative RT-PCR to realize the detection of the quantity of the virus genome RNA in the single cell. The method can be used for researching virus copying on the level of the single cell and the relation between the single cell and a host cell and provides a new method for in-depth research of virus infection cytobiology. The technology for separating and cracking the cells has wide applicability, can be directly applied to the separating pretreatment of other kinds of virus-infected cells or normal cells in order that the method can be also used for the detection of other RNA virus genomes, the copying of normal cell genomes and the research on a transcribed molecular mechanism.

Description

technical field [0001] The invention relates to a single-cell real-time fluorescence quantitative RT-PCR method for detecting foot-and-mouth disease virus genome RNA, which is suitable for studying the replication of foot-and-mouth disease virus at the single-cell level, and then analyzing the mechanism of acute infection and persistent infection of the virus, and exploring the mechanism of Relationship between virus and host cell during infection. Background technique [0002] Foot-and-mouth disease virus (FMDV) belongs to the genus FMDV of the Picornaviridae family. The RNA virus family it belongs to has an important position in medicine and veterinary medicine. The virus mainly causes foot-and-mouth disease in cloven-hoofed animals. The genome consists of a positive-sense RNA strand, containing about 8500 nucleotides, and its replication is via a complementary negative-strand RNA as a template. When the virus infects its sensitive cell lines (such as hamster kidney cell ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 郑从义黄璇李勇屈三甫
Owner 广州誉嘉生物科技有限公司
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