Method of performing one-step, single cell RT-PCR

Patent metadata

Field	Value
Publication number	US-9404924-B2
Application number	US-91164210-A
Country	US
Kind code	B2
Filing date	Oct 25, 2010
Priority date	Dec 4, 2008
Publication date	Aug 2, 2016
Grant date	Aug 2, 2016

Abstract

The invention provides methods for detecting virus production, determining frequency and identity of HIV reservoirs, or evaluating gene expression on a single-cell basis using microengraving and RT-PCR.

First claim

We claim:

1. A method of performing one-step reverse transcription polymerase chain reaction (RT-PCR) with one cell or a few cells, the method comprising steps of:
sealing a plurality of microwells in a microdevice, wherein each microwell in the plurality contains a subnanoliter volume of liquid, one to three cells and reagents suitable to perform cell lysis and RT-PCR, the sealing forming a substantially fluid-tight seal;
heating the sealed microwells such that cell lysis occurs in the sealed microwells; and
subjecting the sealed microwells to thermocycling such that RT-PCR occurs in the microwells, thereby performing one-step RT-PCR with one or a few cells.

2. The method of claim 1 , wherein the step of sealing comprises sealing the microwells to a glass slide.

3. The method of claim 1 , wherein the reagents comprise a lysis reagent, a reverse transcription reagent, a complementary deoxyribonucleic acid (cDNA) amplification reagent, and primers.

4. The method of claim 1 , wherein the reagents comprise detergent, reverse transcriptase, Taq polymerase, and gene-specific probe and primers, wherein the gene specific probe comprises a fluorophore covalently attached to the 5′-end of an oligonucleotide probe and a quencher at the 3′-end of the oligonucleotide probe for real-time PCR quantification.

5. The method of claim 1 , wherein the reagents comprise a ribonuclease (RNase) inhibitor.

6. The method of claim 1 , wherein the reagents further comprise a reference dye 5-carboxy-X-rhodamine (ROX).

7. The method of claim 1 , wherein the microwells are between 10 and 100 μm in diameter.

8. The method of claim 1 , wherein the subnanoliter volume is in a range of 1 pL to 500 pL.

9. The method of claim 1 , further comprising, before said sealing, a step of culturing the cells in the presence of an anti-retroviral compound, and then depositing the cells in the microwells of the microdevice.

10. The method of claim 9 , further comprising, after said depositing and before said sealing, a step of determining by microengraving which cells in the microwells are producing viral peptides, viral fragments, or whole virions;
identifying the cells as virus-producing cells if the cells are determined to be producing viral peptides, viral fragments, or whole virions; and
identifying the cells as non-virus-producing cells if the cells are determined not to be producing viral peptides, viral fragments, or whole virions;
wherein the virus is human immunodeficiency virus (HIV), human T cell leukemia virus (HTLV), herpes simplex virus (HSV) 1, HSV 2, or human endogenous retrovirus (Herv).

11. The method of claim 10 wherein the step of determining which cells produce viral peptides, viral fragments, or whole virions comprises performing steps of:
capturing viral peptides, viral fragments, or whole virions produced by the cells on a glass slide functionalized with capture antibody, and
probing the glass slide with fluorescently-labeled detection antibody.

12. The method of claim 11 , wherein the RT-PCR amplifies an mRNA selected from the group consisting of group-specific antigen (gag) messenger ribonucleic acid (mRNA), polymerase (pol) mRNA, and envelope (env) mRNA.

13. The method of claim 10 , wherein the RT-PCR amplifies an mRNA selected from the group consisting of group-specific antigen (gag) messenger ribonucleic acid (mRNA), polymerase (pol) mRNA, and envelope (env) mRNA.

14. The method of claim 1 , wherein the cells are immune cells.

15. The method of claim 14 , wherein the immune cells are selected from the group consisting of macrophages, monocytes, dendritic cells, and T cells.

16. The method of claim 14 , wherein the immune cells are T cells.

17. The method of claim 1 , wherein the RT-PCR is performed to amplify an mRNA selected from the group consisting of group-specific antigen (gag) messenger ribonucleic acid (mRNA), polymerase (pol) mRNA, and envelope (env) mRNA.

18. The method of claim 1 , wherein the RT-PCR amplifies a viral mRNA.

Assignees

• Love J Christopher
• Gong Yuan
• Massachusetts Inst Technology

Inventors

• Love J Christopher
• Gong Yuan

Classifications

• C12Q1/703

  Viruses associated with AIDS · CPC title

• C12Q2600/158

  Expression markers · CPC title

• G01N33/56983 Primary

  Viruses · CPC title

• C12Q2600/136

  Screening for pharmacological compounds · CPC title