Dataset Information

Implementation of an extraction-free COVID RT-PCR workflow in a pediatric hospital setting.

ABSTRACT:

Background

This study outlines the development, implementation, and impact of a laboratory-developed, extraction-free real-time polymerase chain reaction (RT-PCR) assay as the primary diagnostic test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a pediatric hospital.

Methods

Clinical specimens from both upper and lower respiratory tract sources were validated, including nasopharyngeal aspirates, nasopharyngeal swabs, anterior nares swabs and tracheal aspirates (n = 333 clinical samples). Testing volumes and laboratory turnaround times were then compared before and after implementation to investigate effects of the workflow changes.

Results

Compared to magnetic-bead extraction platforms, extraction-free RT-PCR demonstrated ≥ 95% positive percent agreement and ≥ 97% negative percent agreement across all tested sources. Implementation of this workflow reduced laboratory turnaround time from an average of 8.8 (+/- 5.5) hours to 3.6 (+/- 1.3) hours despite increasing testing volumes (from 1515 to 4884 tests per week over the reported period of testing).

Conclusions

The extraction-free workflow reduced extraction reagent cost for SARS-CoV-2 testing by 97%, shortened sample handling time, and significantly alleviated supply chain scarcities due to the elimination of specialized extraction reagents for routine testing. Overall, this assay is a viable option for laboratories to increase efficiency and navigate reagent shortages for SARS-CoV-2 diagnostic testing.

SUBMITTER: Dumm RE

PROVIDER: S-EPMC8394822 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe speed and sensitivity of real-time polymerase chain reaction (PCR) have made it a popular method for the detection of microbiological agents in both research and clinical specimens. For the detection and genotyping of herpes simplex virus (HSV) in clinical specimens, real-time PCR has proven to be faster, more sensitive and safer than earlier methods which included isolation of the virus in cell culture followed by immunofluorescence microscopy. While PCR-based assays for HSV detection posses clear advantages over these earlier techniques, certain aspects of the PCR method remain onerous. The process of extraction and purification of nucleic acid from clinical specimens prior to PCR is particularly cumbersome. Nucleic acid extraction is expensive, time-consuming and provides a step whereby specimens can become contaminated prior to their analysis. Herein, we investigate the necessity of nucleic acid extraction from swab-based clinical specimens for HSV detection by real-time PCR. We find that nucleic acid extraction is unnecessary for specific and sensitive detection of HSV in clinical specimens using real-time PCR.MethodsProspective (n = 36) and retrospective (n = 21) clinical specimens from various anatomical sites were analyzed for the presence of herpes simplex virus 1 or 2 by real-time PCR using the RealArt HSV 1/2 LC PCR Kit. Specimens were analyzed by PCR both before and following automated nucleic acid extraction. PCR using extracted and unextracted specimens was also compared to cell culture as a means of detecting HSV.ResultsDetection of HSV 1/2 DNA in clinical specimens by real-time PCR did not require that the specimen be subjected to nucleic acid extraction/purification prior to analysis. Each specimen that was detectable by real-time PCR when analyzed in the extracted form was also detectable when analyzed in the unextracted form using the methods herein. The limit of detection of HSV-1 and HSV-2 particles when analyzed in the unextracted form was found to be approximately 17 and 32 virus particles respectively, compared to a sensitivity of 10 copies, for analysis of purified DNA. Omission of the nucleic acid extraction step shortened both the assay time and cost.ConclusionOmission of the nucleic acid extraction step prior to real-time PCR for detection of herpes simplex virus resulted in a more rapid and cost-effective assay, with little impact upon the sensitivity of detection.