Several different pathogenic bacteria, DNA- and RNA viruses and single nucleotide polymorphisms (SNPs) are analysed at the molecular biology laboratory in Skövde.
Since the start of the molecular laboratory in 2002, the numbers of analyses, instruments, and personnel have increased dramatically and the laboratory has doubled its space in the last years. At the moment we are 7 persons in total, performing both the patient analyses and development of new molecular methods.
BDs ProbeTec is used to detect Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), from urine, cervix, urethra, throat and eye samples, and about 27'000 CT and 8'000 NG samples are analysed per year. About half of the samples constitutes of urine. We also analyse Mycoplasma genitalium from genital samples, and therefore we can offer three STI-tests on a single urine sample. All positive sexually transmitted infections (STIs) are confirmed by reanalysing the original sample by CT / NG or by PCR for another specific gene (M. genitalium).
Human and bacterial DNA and viral DNA or RNA is extracted in a Biorobot M48 (Qiagen) with reagents and protocols adapted for each sample type. Thereafter realtime-PCR with SYBR-green, Taqman probe or Molecular beacon probe detection is performed in a Rotorgene instrument (Corbette) or in a LightCycler (Roche). We have two Biorobots, one Lightcycler and three Rotorgene instruments in the laboratory. Last year, in total, we performed, about 20'000 PCR-analyses.
Real-time PCR technology is used for MRSA and VRE screening from overnight broth culture and confirmation of MRSA isolates by nuc/mec and VRE by vanA/vanB gene detection. “Cough-PCR”, i.e. Mycoplasma pneumoniae, Chlamydophila pneumoniae, Bordetella pertussis and Bordetella parapertussis are analysed from a single throat sample.
We also perform 16S rDNA PCR reactions on unknown bacterial isolates before samples are sequenced. In addition, we analyse Calicivirus (norovirus) in faeces or vomits. The most common herpesviruses, HSV-1, HSV-2 and Varicella zoster from blisters are detected by PCR.
Furthermore, a relatively new method for HPV mRNA oncoprotein E6/E7 detection by NASBA technique (Norchip) has been used in our laboratory since the spring 2006.
We also have a method for extracting DNA and detecting representatives from the Mycobacterium tuberculosis complex and the B-globin gene by PCR in paraffin embedded tissues.
Several analyses in the area of molecular genetics are also performed by an MGB based probe assay; an example is the gene marker most commonly causing lactose intolerance in adults, c. 13910C>T, which is detected by this method.
Mutations in the genes for coagulation factors II (prothrombin) and factor V (Leiden), two mutations in the HFE gene associated with hemochromatosis and two polymorphisms in the MTHFR gene (5, 10-methylenetetrahydrofolate reductase) associated with reduced enzyme activity and resulting in potentially increased folate requirements can all be detected using specific PCR-reagents, primers and probes, for each single nucleotide polymorphism (SNP). Last year we performed 6'500 SNP analyses, all on whole blood.
Over the next years we hope to expand our repertoire of molecular analyses even more in all laboratory disciplines including microbiology, clinical chemistry, pathology and human genetics.
References
1. Espy MJ et al. Review. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 2006; 19(1):165-256.
2. Malhotra-Kumar S et al. Minireview. Current trends in rapid diagnostics for methicillin-resistant Staphylococcus aureus and glycopeptide-resistant Enterococcus species. J Clin Micro 2008; 46(5):1577-1587.
3. Janda JM and Abbott SL. Minireview. 16SrRNA gene sequencing for bacterial identifications in the diagnostic laboratory: pluses, perils and pitfalls. J Clin Micro 2007; 45(9):2761-2764.