LNA-substituted degenerate primers improve detection of nitrogenase gene transcription in environmental samples

In order to study the active diazotrophic bacterial community and to capture the majority of its individuals in environmental samples, strategies improving gene detection by increasing sensitivity and efficiency of PCR reactions are highly desirable. Since LNA (locked nucleic acids) modifications might alleviate a low sensitivity and specificity often limiting PCR reactions utilizing degenerate primers, the effect of LNA substituted primers on the detection of nifH transcripts in roots of rice and sugar cane by direct reverse transcription polymerase chain reaction (RT-PCR) was studied. The LNA substitution of the RT primer increased the sensitivity of the RT-PCR up to 26-fold, whereas LNA substitution of the PCR primers decreased specificity. Terminal restriction fragment length polymorphism (T-RFLP) analysis of RT-PCR products showed that LNA substitutions in the RT-primer did not change the pattern of nifH cDNA phylotypes. The use of the LNA-substituted RT-primer allowed the detection of nifH transcripts in sugar cane, where DNA primers alone failed to produce RT-PCR products. These results suggest that similar improvements to PCR detection of nucleic acids can be expected for other environmental samples and genes likewise, when LNA-substituted primers are used.