• As PCR is recognized as an extremely sensitive and specific assay for detecting the presence of pathogen DNA, we have performed multiple molecular detection assays on cell lines for commercial and governmental institutions. Assays which have been utilized include PCR reactions for Mycoplasma genus, Treponema pallidum, Adenovirus, Coxsackie Virus, Cryptococcus neoformans, Cytomegalovirus, Epstein-Barr Virus, Hepatitis G Virus, Herpes Simplex Virus Type 1, Herpes Simplex Virus Type 2, Human Herpes Virus 6 (Variants A and B), Human Herpes Virus 7, Human Herpes Virus 8, Human Papillomavirus, Human T Cell Leukemia Virus, Mycoplasma fermentans, Mycoplasma pneumoniae, Parvovirus, and Varicella-Zoster Virus. When human cell lines are submitted for testing, internal controls specific for human ß-actin or glyceraldehyde-3-phosphate dehydrogenase can be added, if desired.

• MDL also has performed PCR amplification procedures to identify potential unknown bacterial and fungal species that could be contained within DNA extracted from submitted specimens. This is accomplished by utilizing two pairs of bacterial universal primers and one pair of fungal universal primers. The bacterial primers both amplify regions within the 16S ribosomal RNA gene, while the fungal universal primers are capable of amplifying a region within the ITS gene. If a sequence is amplified, it can be purified and sequenced by traditional dideoxynucleotide termination technology or an in-house developed Pyrosequencing assay; the heterogenous stretch of DNA between the primer locations can identify the exact species of the detected bacteria or fungus.

• Real-time PCR assays on the Rotor Gene 3000 platform have been developed and validated for a multitude of clinically relevant pathogens including Neisseria gonhorrhea, Chlamydia pneumoniae, Trichomonas vaginalis, Haemophilus ducreyi, Treponema pallidum, Aspergillus fumigatus, Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, and West Nile Virus. Pyrosequencing technology has permitted the sequencing of amplicons for verification as authentic organisms.

• Pyrosequencing assay development is also an area of expertise at MDL. Assays have been designed and tested to explore antifungal resistance of fungal pathogens such as Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans.

• A Mycoplasma-like tissue culture contaminant was provided to MDL for species identification. PCR amplification utilizing Mycoplasma genus specific primers and DNA sequencing revealed the organism to have 97% sequence homology with Acholeplasma laidlawii. A recent report detailed the presence of A. laidlawii in 6% of the Mycoplasma contamination found from examining 495 cell cultures PCR assays, microbiological culture methods, and DNA-RNA hybridizations.

• MDL offers clinical diagnostic testing for the causative agent of Rocky Mountain Spotted Fever, Rickettsia rickettsii by PCR. Historically, the bacteria were purchased and extracted for use as a positive control but due to the implementation of stricter regulations due to bioterrorism concerns, this was no longer possible. MDL subcloned a portion of the R. rickettsii genome as a solution. Depicted below are PCR amplifications from the vector (diluted from 1:1 to 1:10,000,000), authentic R. rickettsii DNA, and a negative control.

• Formalin-fixed epithelial dysplasia tissue samples were provided to MDL embedded in paraffin blocks. Sections were excised and extracted to yield purified DNA. PCR amplification and DNA sequencing were utilized for human papillomavirus (HPV) detection and genotyping. ß-actin was also amplified as a control from each sample.

• One project had been prescribing probiotic supplements for intestinal disorders. MDL performed molecular authentication by PCR methodologies and DNA sequencing for the presence of the organisms in the Table at right, which were stated by the manufacturer to be present in the probiotics.

• Due to the large quantity of agarose gels that are used at MDL on a daily basis, it was an economical decision to subclone our own molecular weight ladder, shown in the second lane at right. Sections of the Borrelia burgdorferi genome were selected, amplified by PCR, and subcloned into a bacterial cloning vector. Digestion of large scale plasmid preparations of this vector with the restriction enzyme HindIII created a molecular weight ladder with the following sizes in nucleotides from the top: 2743, 980, 752, 650 (double intensity), 552, 395, 300, 198, and 104. The first lane of the figure at right is a commercially available restriction ladder. This cost-savings step reduced our expenditure for this consumable product by 98% and now saves MDL over $25,000 annually.

• Atherosclerotic plaques were subjected to DNA analysis of pathogens to determine the prevalency of Chlamydia pneumoniae, Helicobacter pylori, Mycoplasma, Epstein-Barr Virus (EBV), and Cytomegalovirus (CMV). As a control, ß-actin was amplified and qualitatively displayed.

• The most significant software development effort currently underway is TRIM - Test Requisition Information Management system. This cutting-edge, state-of-the-art system integrates and manages all aspects of the test requisition process. The clinical component of TRIM includes managing diagnostic details and workflow process, PCR specimen list and reagent calculation generation, results consolidation, reporting and clinical statistical analysis. The business component of TRIM manages billing, reporting and various business and monetary analysis and statistical reports.

Each project is highly individualized. For consultation, please contact:

Dr. Martin E. Adelson
madelson@mdlab.com
877.269.0090

Detailed proposals and itemized descriptions and prices will be provided.