Method for the specific detection of low abundance RNA species in a biological sample

1. A method for the detection of low abundance target RNA species in a biological sample, comprising the steps of:

(a) reverse transcription of RNA into cDNA using a reverse transcriptase (RT), wherein step (a) is performed under hotstart conditions;

(b) inactivation of the RT; and

(c) detection of the target cDNA by polymerase chain reaction (PCR),

wherein the biological sample has a large background of cellular components and a large background of total RNA, and wherein the RT is a recombinant Thermus thermophilus DNA polymerase (rTth-pol).

2. The method of claim 1 , further comprising after step (a) the step of amplifying the target cDNA using the RT of step (a).

3. The method of claim 1 , wherein the RT is inactivated in step (b) by a method selected from the group consisting of heat treatment, treatment with phenol, treatment with proteinase K, and treatment with a chaotropic agent.

4. The method of claim 3 , wherein the RT is inactivated by treatment with a chaotropic agent, further comprising after step (b) the step of removing the chaotropic agent.

5. The method of claim 4 , wherein the chaotropic agent is selected from the group consisting of urea, lithium perchlorate, and a guanidinium salt.

6. The method of claim 1 , wherein the polymerase chain reaction (PCR) of step (c) is a real-time PCR.

7. The method of claim 1 , wherein the target RNA is derived from a mycoplasma.

8. The method of claim 7 , wherein the mycoplasma is selected from the group, consisting of Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma hyorhinis, Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma synoviae , and Mycoplasma orale.

9. The method of claim 7 , wherein the target RNA is 16S ribosomal RNA.

10. A method for the detection of a mycoplasma contamination in a biological sample, comprising the steps of:

(a) extraction of total RNA from the biological sample;

(b) reverse transcription of RNA into cDNA using a reverse transcriptase (RT);

(c) amplification of cDNA derived from mycoplasma 16S ribosomal RNA (16S rRNA) by polymerase chain reaction (PCR) using the RT of step (b);

(d) inactivation of the RT by addition of a chaotropic agent;

(e) removal of the chaotropic agent from the reaction mixture; and

(f) detection of cDNA derived from mycoplasma 16S rRNA by polymerase chain reaction (PCR),

wherein the RT is a recombinant Thermus thermnophilus DNA polymerase (rTTh-pol).

11. The method of claim 10 , wherein step (b) is performed under hotstart conditions.

12. The method of claim 10 , wherein the chaotropic agent is selected from the group consisting of urea, lithium perchlorate, and a guanidinium salt.

13. The method of claim 10 , wherein the polymerase chain reaction (PCR) of step (f) is a real-time PCR.

14. The method of claim 10 , wherein the mycoplasma is selected from the group, consisting of Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma hyorhinis, Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma synoviae , and Mycoplasma orale.