Purifying bacterial DNA from contaminated food samples

Contaminated food is becoming an increasing bacteria are the most common cause of food-borne illnesses. Lambertz et al. at the National Food Administration in Finland describe a sensitive and specific real-time PCR assay to detect the food-borne pathogen Yersinia pseudotuberculosis. Traditional culture-based methods are limited by the low isolation rate of this Gram-negative bacterium in naturally contaminated samples.  Using the MasterPure™ Complete DNA and RNA Purification Kit, the researchers isolated DNA from 25 food samples including mixed salad, minced meat, nonpasteurized milk, carrots, turnips, cabbage, lettuce, onion, pumpkin, and tomatoes. They developed TaqMan® qPCR assays using gene-specific probes to detect the Y. pseudotuberculosis ail gene, and distinguish it from related Y. enterocolitica serotypes. Whereas 6 of the analyzed food samples were positive for Y. pseudotuberculosis as indicated by PCR, all 25 were negative when analyzed by the traditional culture method.

Lambertz, S. et al. (2008). TaqMan-Based Real-Time PCR Method for Detection of Yersinia pseudotuberculosis in Food Applied and Environmental Microbiology, 74 (20), 6465-6469 DOI: 10.1128/AEM.01459-08

Structural differences between Marburg and Ebola viruses

Ebola virus (EBOV) and Marburg virus (MARV) are related pathogens that cause hemorrhagic fevers. In many cases, viral infections are fatal. Both viruses are native to Africa where outbreaks have been occurring for decades. There is no effective therapy for the hemorrhagic fevers caused by these viruses. EBOV and MARV are in the same taxonomic family and are structurally identical; however, they elicit different antibodies. Enterlein et al.* used the AmpliScribe™ T7 High Yield Transcription Kit to investigate differences in RNA secondary structures between MARV and EBOV. They analyzed the structure of the MARV 3’-noncoding region and its influence on VP30. VP30 is an RNA binding protein which acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription.

The researchers choose the AmpliScribe system because it can efficiently transcribe RNA from limited amounts of DNA (as low as 1 ng). The AmpliScribe High Yield Transcription Kits can produce >20-fold more full-length RNA (both short and long transcripts) than conventional in vitro transcription reactions.

Enterlein, S. et al. (2009). The Marburg Virus 3' Noncoding Region Structurally and Functionally Differs from That of Ebola Virus Journal of Virology, 83 (9), 4508-4519 DOI: 10.1128/JVI.02429-08

Visit Epicentre at ASM 2010 in San Diego, CA

We’re gearing up for our annual pilgrimage to the American Society for Microbiology (ASM) General Meeting, May 23-27. Searching the abstracts, we noted many posters and presentations citing Epicentre’s unique products for molecular biology research. Posters and student seminars have been presented at previous ASM meetings featuring EZ-Tn5™ transposon mutagenesis, large-insert cloning with CopyControl™ products, PCR using the renowned FailSafe™ PCR System, DNA purification from a variety of sources using the MasterPure™ kits, and many other Epicentre products.

For those of you attending ASM 2010, you can search the abstracts for topics of interest and citations of Epicentre products.  Also, visit our exhibit (#122) to learn more about:

• Creating metagenomic libraries from water, soil, compost, and air samples;
• Nextera™ Sample Prep Technology for next-generation sequencing of microbial and metagenomic DNA libraries;
• In vivo and in vitro transposition systems for DNA recombination and engineering.

Technical support and marketing staff will be present to answer your questions and discuss our new products. And, of course, we’ll have our booth stocked with ever-popular giveaway items!

We hope to see you at ASM 2010! Feel free to leave a comment and let us know if you’re attending.

Isolation of RNA from breast cancer FFPE tissues

In order to develop guidelines for clinical diagnostic tests using gene expression profiling based on qRT-PCR analyses of formalin-fixed, paraffin-embedded (FFPE) tissues, Sánchez-Navarro et al.* compared the performance of different normalization strategies in the correlation of quantitative data between fresh-frozen (FF) and FFPE tissues.  A significant challenge to expression analysis of FFPE samples is the substantial degradation of RNA extracted from these tissues, resulting in a shift in raw CT values compared to FF tissues. However, the authors demonstrate that proper normalization of the expression data can compensate of the effects of RNA degradation. The authors used the MasterPure™ RNA Purification Kit to isolate RNA from FFPE tissue slices and examined expression levels of reference and breast cancer prognosis–related genes using TaqMan® low-density arrays. Based on their analysis, they make recommendations for proper normalization strategies when using FFPE samples in qRT-PCR analysis. The authors conclude:

Nevertheless, careful selection of candidate biomarkers should be made: those genes that show no correlation between FF and FFPE should not be included in molecular tests for clinical use based in FFPE samples. Moreover, in order to guarantee reliable results in gene expression measurements, we strongly encourage performing preliminary studies, with the aim of discarding noncorrelated genes.

Sánchez-Navarro, I. et al. (2010). Comparison of gene expression profiling by reverse-transcription quantitative PCR between fresh frozen and formalin-fixed, paraffin-embedded breast cancer tissues BioTechniques, 48 (May 2010), 389-397 : 10.2144/000113388

Nextera™ library fragment size range

Another common question regarding our Nextera™DNA Sample Prep Kits for next-generation sequencing:

The Nextera library fragments are too small. Is there a better way to control the DNA fragment size?

Nextera DNA Sample Prep kits include two buffers, Low Molecular Weight (LMW) and High Molecular Weight (HMW), for generating two size classes. In general, with the Nextera Illumina-Compatible Kit, the LMW buffer will produce fragments from 150 to 600 bp (peak around 200 bp). The HMW buffer will produce fragments from 175 to 700 bp (peak around 250 bp). The final fragment size includes approximately 100 bp of adaptor/transposon end sequence, so the actual genomic fragment is smaller than the apparent MW of the library. It is normal for the fragment MW to vary slightly. This is commonly a result of DNA type, quality, and purification and quantitation methods used.

The quality of the starting DNA is critical. Contaminants such as protein and RNA may inhibit the Nextera tagmentation reaction if present in the DNA preparation. Therefore, it is important to start with highly pure  DNA. We also recommend using HMW buffer if small fragment size is an issue. The current protocol has been developed with 50 ng of DNA. If the fragments are too small, we recommend adding slightly more DNA. Changing the temperature or reaction time is not a robust or reproducible way to control fragment size. Finally, if a narrow MW distribution is required, it may be necessary to perform a size-selection step (gel, AMPure® beads, SPRIWorks®, etc.) after the limited-cycle PCR.

Transposon mutagenesis identifies a novel toxin regulatory locus in Clostridium perfringens

Over the years, we’ve had many inquiries about using the EZ-Tn5™ Transposomes on biologically interesting but difficult-to-mutate bacteria--usually Gram-positive bacteria that have poorly understood genetics and are difficult to transform with foreign DNA. As time has progressed, some of the difficulties of using EZ-Tn5 Transposomes have been overcome. An example of such success was recently reported by Vidal et al.* regarding the use of a custom EZ-Tn5 Transposome that confers tetracycline resistance to its targeted host cell. Clostridium perfringens is a Gram-positive, obligate spore-forming anaerobe. Its growth characteristics pose considerable obstacles to the use of transposon mutagenesis in the search for genes associated with virulence factors.

The authors used the pMOD-2 Transposon Construction Vector to build an erythromycin-resistance transposon using the erm gene from the E. coli-C. perfringens shuttle vector pJIR751. The Transposome complex was built using standard procedures and transformed using a Biorad Gene Pulser™ electroporator. Using the EZ-Tn5 system, the researchers were able to locate a mutant that disabled the toxin-13 regulatory locus (determined to be similar to the Agr locus of Staphylococcus aureus). They also reported that the generation of mutant Clostridia using the EZ-Tn5 Transposomes was much more efficient than other random mutagenesis methods.

If you're considering the EZ-Tn5 system for bacterial mutagenesis, visit the EZ-Tn5 Transposomes citation page for information on your species of interest.

*Vidal, J. et al. (2009). Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13 PLoS ONE, 4 (7) DOI: 10.1371/journal.pone.0006232