A rapid and sensitive system for recovery of nucleic acids from Mycobacteria sp. on archived glass slides

Abstract

BackgroundThe field of diagnostics continues to advance rapidly with a variety of novel approaches, mainly dependent upon high technology platforms. Nonetheless much diagnosis, particularly in developing countries, still relies upon traditional methods such as microscopy. Biological material, particularly nucleic acids, on archived glass slides is a potential source of useful information both for diagnostic and epidemiological purposes. There are significant challenges faced when examining archived samples in order that an adequate amount of amplifiable DNA can be obtained. Herein, we describe a model system to detect low numbers of bacterial cells isolated from glass slides using (laser capture microscopy) LCM coupled with PCR amplification of a suitable target.ResultsMycobacterium smegmatis was used as a model organism to provide a proof of principle for a method to recover bacteria from a stained sample on a glass slide using a laser capture system. Ziehl-Neelsen (ZN) stained cells were excised and catapulted into tubes. Recovered cells were subjected to DNA extraction and pre-amplified with multiple displacement amplification (MDA). This system allowed a minimum of 30 catapulted cells to be detected following a nested real-time PCR assay, using rpoB specific primers. The combination of MDA and nested real-time PCR resulted in a 30-fold increase in sensitivity for the detection of low numbers of cells isolated using LCM.ConclusionsThis study highlights the potential of LCM coupled with MDA as a tool to improve the recovery of amplifiable nucleic acids from archived glass slides. The inclusion of the MDA step was essential to enable downstream amplification. This platform should be broadly applicable to a variety of diagnostic applications and we have used it as a proof of principle with a Mycobacterium sp. model system.

Isolation of DNA from spots of old microscopic glass slides with mini column isolation kit for molecular analysis

“…The slides can be fixed to inactivate the pathogens and fixed slides can be stored for many years as in this method, very old, fixed slides are used over a long period of time. [13,14,16] Many pathological departments and microbiological departments have old, fixed glass slides; hence DNA can be isolated from them with our method to conduct the molecular studies to see the presence of a pathogen as well as genetic studies. Till we rarely found that there was any problem with any of pathogen DNA isolation from slides, however we faced some issues with some mounted histological fixed slides with human tissues, but for RNA isolation.…”

“…To this mini column, add 100 µl from microtube called EL was added. 16. This was kept at room temperature for 2 minutes.…”

Draft Genome Sequence of Mycolicibacterium smegmatis VKM Ac-1171 Contains Full Set of Sterol Catabolic Genes

“…Recently, Mycobacterium smegmatis and closely related fast-growing species have been reclassified as Mycolicibacterium based on their phylogenomic differences from the “tuberculosis-like” clade ( 2 , 3 ). The strain has previously been used in various studies as a nonpathogenic model microorganism ( 4 – 9 ), to validate the DNA isolation method ( 10 ), but its genome sequence was hitherto unreported. Here, we present a draft genome of M. smegmatis VKM Ac-1171.…”

Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides

“…Recently, specific PCR assays have been developed for use on cytology slides for the detection of neoplastic and infectious diseases such as mycobacteriosis and leishmaniasis. 14,16,24 Cytology slides may be advantageous compared to FFPE tissues for molecular testing because they can be prepared more rapidly and often without the need for anesthesia; furthermore, the fixation method is more gentle with less degradation of DNA, and there are fewer chances for contamination during processing steps. 1,11,28 This study aimed to evaluate the suitability of stained cytology slides as samples for direct panfungal PCRsq.…”