Real time pcr in Rotor gene 6000- not getting positive results - (Mar/11/2008 )

I have been trying to diagnose tuberculosis positive cases by real time pcr from hospital samples using Rotor gene 6000 of Corvette Research and using standard kits. Since I work in one of the most highly endemic zones for TB in India I expected a large number of positive results from apparently AFB negative samples. But to my utter surprise, all samples seem to be negative which is just not possible. Sometimes even AFB +ve samples are not showing amplification.

I strictly followed the kitmakers instructions and also tried the standard modifications suggested for TB in the literature like heatshock, decontamination, lysozyme treatment etc. depending upon the sample type, but nothing seems to be working, even the kitmaker was unable to suggest a suitable remedy.

I will be very grateful if anyone could please suggest what other steps should I check, for I am extremely keen to get it right.

tkb

Your kit maker should have some sort of positive control sample, which you can use to verify that the kit reagents and machine are functioning properly. It is very hard to suggest more without knowing the kit contents and protocol. Are there storage or shipping problems with the kit contents?