Universal Transport Medium (UTM – RT) With Flocked Swabs and Real – Time PCR Detects Viral Nucleic Acids

ABSTRACT:

Background:

Nucleic acid amplification assays are becoming widely used for the laboratory diagnosis of viral infections. A versatile transport medium and collection system, effective for all laboratory technologies is necessary.

Objective:

To compare a room temperature universal transport medium [UTM‐RT] and flocked swabs [FS] (Copan) to M4‐RT (Remel) transport medium and Micro Gent Stainless Steel/Plastic swabs (MG) for the detection of HSV1, HSV 2, VZV, CMV, Influenza A, Influenza B, and Echovirus 11 in contrived specimens. To test clinical samples collected into UTM‐RT with FS in RT‐PCR and by DFA/culture.

Methods:

Laboratory strains of HSV1, HSV 2, VZV, CMV, Influenza A, Influenza B, and Echovirus 11 were serially diluted ten fold in UTM‐RT and M4‐RT from 10‐1 to 10‐10. One ml of each dilution for both media was aliquoted into sterile vials (contrived specimens). A swab, provided with each transport media, was added to the respective vials. Suspensions were vortexed, swabs were removed, 0.2 ml was inoculated into 2 shell vial cell cultures each. For RT‐PCR nucleic acid was extracted from 0.2ml using a manual magnetic silica method and extracted nucleic acids were tested in duplicate in the LightCycler using artus RealArttm‐LC PCR kits (Qiagen). A total of 48 culture positive clinical samples collected by FS into UTM‐RT were tested by RT‐PCR, after freezing and thawing. Infected cell cultures were stained using FITC monoclonal antibodies.

Results:

Endpoints of detection of the contrived specimens were equivalent for the combination of FS with UTM‐RT to the MG in M4‐RT for HSV1 and 2, CMV, VZV and Influenza A. Endpoint determinations were enhanced 10‐fold for Influenza B and Echovirus 11 by the FS/UTM‐RT. The artus LC real‐time PCR was more sensitive than shell vial culture with both swab/transport systems with contrived specimens. RT‐PCR detected 46 /48 of clinical positives from UTM‐RT.

Conclusions:

The FS/UTM‐RT could serve as a universal system for detecting viruses by real time PCR.