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Mycoplasma orale has a sequence similar to the insertion-like sequence of M. fermentans
S E Ditty1, M A Connolly, B J Li
1Department of Infectious and Parasitic Diseases Pathology, American Registry of Pathology, Washington, DC 20306, USA.
Abstract:
Polymerase chain reaction (PCR) using the Mycoplasma fermentans insertion sequence-like element (ISLE) RW primer set amplifies DNA from Mycoplasma orale when more than 1 ng is present in the reaction tube. In this study, amplified products from 11 different clinical isolates and the ATCC prototype of M. orale were sequenced and compared to 206 bp amplicons from eight isolates and the ATCC strain of M. fermentans. The nucleotide sequences of the amplified M. orale products had high sequence homology (88-92%) to those from M. fermentans, but differed at several key positions. The M. orale products contained a DraI restriction enzyme site not found in any of the M. fermentans amplified products. Consistent with this finding, the PCR products from M. orale were digested by DraI while the PCR products from M. fermentans were resistant to DraI digestion. The results suggest that M. orale may carry a similar IS-like element that complicates but does not negate using the ISLE PCR assay designed to detect M. fermentans. It appears possible for the RW primers to amplify M. orale if the mycoplasmas are present at higher concentrations. The amplified products can be differentiated from those from M. fermentans by a rapid DraI restriction endonuclease digestion or by Southern blot analysis using the RW006 internal probe under highly stringent conditions.
Insights
The Mycoplasma fermentans insertion sequence-like element (ISLE) PCR assay can amplify Mycoplasma orale DNA. Amplified products show sequence homology but can be differentiated using DraI restriction digestion.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Mycoplasma fermentans is a pathogen detected using PCR assays targeting its insertion sequence-like element (ISLE).
- The specificity of the ISLE PCR assay for M. fermentans needs evaluation against related species like Mycoplasma orale.
Purpose of the Study:
- To investigate the cross-reactivity of the ISLE PCR assay with Mycoplasma orale.
- To determine if M. orale DNA can be amplified by the M. fermentans-specific ISLE PCR assay.
- To identify methods for differentiating amplified products from M. orale and M. fermentans.
Main Methods:
- DNA amplification using Polymerase Chain Reaction (PCR) with RW primers targeting ISLE.
- Sequencing of amplified DNA products from M. orale and M. fermentans isolates.
- Restriction fragment length polymorphism (RFLP) analysis using DraI enzyme.
- Southern blot analysis with RW006 probe under stringent conditions.
Main Results:
- The ISLE PCR assay amplified M. orale DNA when concentrations exceeded 1 ng.
- Sequencing revealed 88-92% homology between amplified M. orale and M. fermentans DNA, with key differentiating positions.
- M. orale PCR products contained a DraI restriction site absent in M. fermentans products, allowing differentiation via DraI digestion.
- Southern blot analysis also differentiated the amplified products.
Conclusions:
- The ISLE PCR assay designed for M. fermentans can amplify M. orale DNA, particularly at higher concentrations.
- This cross-reactivity complicates, but does not invalidate, the assay's use.
- DraI restriction digestion or stringent Southern blot analysis can reliably distinguish between M. orale and M. fermentans PCR products.