Method for the direct detection of diagnostic RNA
Methods for the direct detection of diagnostic target RNA have been developed, which obviate the need for time consuming RNA purification and isolation procedures.
1. A method for the direct detection of a diagnostic target RNA from a bacteria, comprising the steps of:
a) providing a sample comprising at least a portion of at least one bacterial cell, said portion of said cell comprising a diagnostic target RNA;
b) contacting the sample with an RT-PCR composition to form an RT-PCR reaction mixture;
c) thermocycling the RT-PCR reaction mixture, whereby a diagnostic target DNA product is amplified; and
d) detecting the amplified diagnostic target DNA product of (c), with the proviso that no step includes RNA purification and wherein the sample of (a) does not contain an exogenously added RNase inhibitor.
2. A method for the direct detection of a diagnostic target RNA from a bacteria, comprising the steps of:
a) providing a sample comprising at least a portion of at least one bacterial cell, said portion of said cell comprising a diagnostic target RNA;
b) contacting the sample with an RT reaction composition to form an RT reaction mixture under conditions, whereby a diagnostic DNA product is transcribed from the RNA;
c) detecting the diagnostic target DNA product, with the proviso that no step includes RNA purification and wherein the sample of (a) does not contain an exogenously added RNase inhibitor.
3. A method according to claim 2 , further comprising after step (b), the steps of:
contacting the diagnostic DNA product with a PCR composition to result in a PCR reaction mixture; and
thermocycling the resultant PCR reaction mixture, whereby the diagnostic target DNA product is amplified.
4. A method according to claim 2 , wherein the detecting comprises incorporating or attaching a label to the diagnostic target DNA product.
5. A method according to claim 3 , wherein the label is selected from the group consisting of fluorescent moieties, chemiluminescent moieties, particles, enzymes, radioactive tags and light emitting moieties.
6. A method according to any of claims 1 , 2 or 3 , further comprising contacting the sample with a lysing agent.
7. A method according to claim 6 , wherein the lysing agent is selected from the group consisting of a lysis buffer and high temperature.
8. A method according to claim 6 , wherein the lysing agent is selected from the group consisting of a chemical, an enzyme and physical shearing.
9. A method according to claim 8 , wherein the enzyme is selected from the group consisting of lysozyme, glucolase, zymolose, lyticase and viral endolysins.
10. A method according to claim 9 , wherein the viral endolysin is selected from the group consisting of endolysins from bacteriophages or prophages and combinations of these.
11. A method according to claim 9 , wherein the viral endolysin is selected from the group consisting of endolysins from Listeria bacteriophages A118 and PLY118, endolysins from bacteriophage PM2, endolysins from the B. subtilis bacteriophage PBSX, endolysins from Lactobacillus prophages Lj928, Lj965 and bacteriophage Phiadh, endolysin (Cpl-1) from the Streptococcus pneumoniae bacteriophage Cp-1, the bifunctional peptidoglycan lysin of Streptococcus agalactiae bacteriophage B30, two-component cell lysis genes holWMY and lysWMY of the Staphylococcus warneri M phage varphiWMY and combinations of these.
12. A method according to claim 8 , wherein the physical shearing is produced by zirconia-silica beads or a French press.
13. A method according to any of claims 1 , 2 or 3 , wherein the sample is heated to a temperature in the range of about 50° C. to less than or about 100° C.
14. A method according to any of claims 1 , 2 or 3 , wherein the diagnostic target RNA is selected from the group consisting of mRNAs, rRNAs, tRNAs and small RNA species.
15. A method according to claim 14 , wherein the rRNAs are selected from the group consisting of 5S RNA, 16S RNA, and 23S RNA.
16. A method according to any of claims 1 , 2 or 3 , wherein the at least one bacterial cell is a member of a genus selected from the group consisting of Listeria, Escherichia, Salmonella, Campylobacter, Clostridium, Helicobacter, Mycobacterium, Staphylococcus, Camplobacter, Enterococcus, Bacillus, Neisseria, Shigella, Streptococcus, Vibrio, Yersinia, Bordetella, Borrelia , and Pseudomonas.
17. A method according to any of claims 1 , 2 , 3 or 4 , wherein the sample is selected from the group consisting of food, meat, flesh products, dairy products, fruit, ground beef and combinations of these.
18. A method according to claims 1 , 2 , 3 , or 4 , wherein the sample is selected from the group consisting of samples taken from a food processing context and samples taken from a medical context.