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An undergraduate laboratory experiment with real-world applications: Utilizing templateless polymerase chain reaction and real-time polymerase chain reaction to test for SARS-CoV-2 RNA.

ABSTRACT: We present a course-embedded undergraduate research module that involves real-time polymerase chain reaction testing for the presence of SARS-CoV-2 in environmental samples. A positive control RNA was constructed and two RNA extraction methods were compared and a range of primers were available to compare. Using a combination of published protocols, we assembled a successful project that illustrated a topical exercise similar to real-world assay development. The exercise is aimed at upper-level undergraduates and requires 3 weeks of laboratory periods. The students were able to design and test experimental protocols, while learning about RNA detection. This project could be utilized in upper-level classes including molecular biology, biochemistry, biotechnology, or for independent research projects.

SUBMITTER: Crane J

PROVIDER: S-EPMC9011848 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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An undergraduate laboratory experiment with real-world applications: Utilizing templateless polymerase chain reaction and real-time polymerase chain reaction to test for SARS-CoV-2 RNA.

Crane Julia J Page Shallee T ST

Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology 20211204 1

We present a course-embedded undergraduate research module that involves real-time polymerase chain reaction testing for the presence of SARS-CoV-2 in environmental samples. A positive control RNA was constructed and two RNA extraction methods were compared and a range of primers were available to compare. Using a combination of published protocols, we assembled a successful project that illustrated a topical exercise similar to real-world assay development. The exercise is aimed at upper-level ...[more]

PMID: 34862844

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Project description:Background and aimDifferent polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and methodsOne hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.ResultsTaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.ConclusionA cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

Dataset Information

An undergraduate laboratory experiment with real-world applications: Utilizing templateless polymerase chain reaction and real-time polymerase chain reaction to test for SARS-CoV-2 RNA.

Publications

An undergraduate laboratory experiment with real-world applications: Utilizing templateless polymerase chain reaction and real-time polymerase chain reaction to test for SARS-CoV-2 RNA.

Similar Datasets

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