Real-Time PCR Detects Viral Nucleic Acids in Universal Transport Medium (UTM-RT) with Flocked Swabs S. CASTRICIANO*, A. PETRICH, M. SMIEJA and M.A. CHERNESKY Departments of Pathology & Molecular Medicine, and Paediatrics McMaster University, St. Joseph s s Healthcare, Hamilton, ON
Background: Nucleic acid amplification assays are becoming widely used for laboratory diagnosis of viral infections. A versatile transport medium and collection system, effective for all laboratory technologies is necessary.
Objectives: To compare two room temperature universal transport media systems for the detection of viral nucleic acids in contrived specimens by real time PCR. -The UTM-RT and flocked swabs (Copan) -The M4-RT (Remel) transport medium and MicroGent stainless steel/plastic swabs. To test the ability to detect viral nucleic acids by real time PCR in DFA/culture positive clinical specimens collected into UTM-RT with flocked swabs.
UTM-RT and Flocked Swab (Copan Diagnostics Inc.) M4-RT and steel/plastic swabs (Remel)
Methods: Contrived specimens were prepared in both transport systems using laboratory strains of: HSV1 HSV 2 VZV CMV Influenza A Influenza B Echovirus 11
Methods: 48 frozen positive clinical specimens, collected by flocked swabs and placed into UTM-RT, were used in this study. The specimens used were positive by culture and identification with type specific FITC monoclonal antibodies. Contrived and clinical specimens were tested with the artus RealArt tm -LC PCR kits (Qiagen( Qiagen).
Contrived Specimen Preparation: Each virus was serially diluted in UTM-RT and M4- RT from -1 to -. One ml of each virus dilution was used for both media and was aliquoted into sterile vials. A flocked swab was added to each virus dilution in UTM-RT A steel/plastic swab was added to each virus dilution in M4-RT
Contrived Specimens Culture All contrived specimens were vortexed, and the swabs were removed prior to testing. 0.2 ml of each contrived specimens was inoculated into 2 shell vial cell cultures. R-Mix cells for the Influenza A, B, and Echo 11 H and V Mix cells for HSV1 and 2, CMV, VZV.
RT-PCR: 0.2 ml volume of all contrived and frozen clinical specimens was used for extraction of nucleic acid A manual magnetic silica method was used for extraction of both RNA and DNA. 5 µl l of extracted nucleic acid from each sample was tested in duplicate in the LightCycler using artus RealArt tm -LC PCR kits (Qiagen).
Results: The contrived specimens prepared with flocked swabs in UTM-RT and the steel/plastic swabs in M4-RT had equivalent PCR endpoints of detection for: Influenza A, HSV1 and 2, CMV, and VZV. Influenza A 40 35 Crossing Points 30 25 20 15 5 UTM-RT M4-RT 0-1 -2-3 -4-5 -6-7 Dilutions
Herpes Simplex 1 Cytomegalovirus 35 30 Crossing Points 30 25 20 15 5 UTM-RT M4-RT Crossing Points 25 20 15 5 UTM-RT M4-RT 0-4 -5-6 -7-8 Dilutions 0-3 -4-5 -6-7 -8-9 Dilutions Herpes Simplex 2 Varicella Zoster Virus 35 35 Crossing Points 30 25 20 15 5 0-4 -5-6 -7-8 UTM- RT M4- RT Crossing Points 30 25 20 15 5 0-2 -3-4 -5-6 -7-8 UTM-RT M4-RT Dilutions Dilutions
Echovirus 11 45 40 Crossing Points 35 30 25 20 15 5 0-5 -6-7 -8-9 Dilutions UTM-RT M4-RT The contrived specimens prepared with flocked swabs in UTM-RT enhanced PCR endpoint determinations -fold for Influenza B and Echovirus 11. Influenza B 35 30 Crossing Points 25 20 15 5 UTM-RT M4-RT 0-1 -2-3 -4-5 Dilutions
Results: The artus LC real-time PCR was more sensitive than shell vial culture with both swab/transport systems with contrived specimens. The contrived specimens prepared with flocked swabs/ UTM-RT demonstrated enhanced PCR endpoint determinations -fold for Influenza B and Echovirus 11. RT-PCR detected 46 /48 of clinical positives from UTM- RT.
Contrived Specimens Culture/PCR Results Viruses FS/UTM-RT MG/M4-RT Culture PCR Culture PCR Influenza A -5-6 -5-6 Influenza B -3-4 -2-3 Echo 11-6 -8-5 -7 HSV 1-5 -7-5 -7 HSV 2-5 -7-4 -7 VZV -5-7 -4-7 CMV -6-8 -5-8
Clinical Samples PCR Results: Positive Viruses Clinical samples (collected in UTM-RT with Flocked) swabs VZV Echo11 Influenza A Influenza B CMV Total RT-PCR +/- 9/0 6/2* 13/0 /0 /0 46 Culture + 9 8 13 48 * Due to freezing and thawing.
Conclusions: The FS/UTM-RT could serve as a universal system for detecting viruses by real time PCR in combination with the artus RealArt tm -LC PCR kits The contrived specimens prepared with flocked swabs in UTM-RT enhanced PCR endpoint determinations-fold for Influenza B and Echovirus 11. PCR is more sensitive then culture by 1 to 2 log dilution for most of the viruses.