Sample Preparation for PCR

Lee, Margie D.; Fairchild, Amanda J.

doi:10.1007/0-387-31702-3_3

Cited by 9 publications

(3 citation statements)

References 27 publications

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“…PCR inhibitors may be lipids, proteins, enzymes, chemical additives, fiber, other bacteria, or pH (Lee & Fairchild, 2006). The size of the IAC used in the present study was smaller than the target.…”

Section: Discussionmentioning

confidence: 96%

Real-time reverse-transcriptase PCR for Salmonella Typhimurium detection from lettuce and tomatoes

Miller

Davidson

D’Souza

2011

LWT - Food Science and Technology

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Section: Discussionmentioning

confidence: 96%

Real-time reverse-transcriptase PCR for Salmonella Typhimurium detection from lettuce and tomatoes

Miller

Davidson

D’Souza

2011

LWT - Food Science and Technology

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“…PCR inhibitors may be lipids, proteins, enzymes, chemical additives, fiber, other bacteria, or pH (Lee et al, 2006). Therefore, we developed an RNA IAC.…”

Section: Discussionmentioning

confidence: 99%

Development of asigDE-Based Real-Time Reverse-Transcriptase PCR for the Detection of ViableSalmonella enterica

Zhou

Yang

Zhou

et al. 2014

Foodborne Pathogens and Disease

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Salmonella is the most common cause of bacterial food poisoning in humans worldwide. Thus, rapid and reliable methods for the detection of this pathogen are required. Real-time reverse-transcriptase polymerase chain reaction (rt-RT-PCR), which detects the presence of mRNA (shorter half-life than DNA) has shown great potential for detecting viable pathogens. We recently identified a few new potential specific DNA sequences for Salmonella enterica using a comparative genomics method (Chen et al., 2010). In the present study, we examined the expression of the Salmonella-specific sigDE operon (encoding invasion proteins within the pathogenicity island 5) under typical growth conditions to determine whether sigDE could be a useful viability marker for the bacterium. We then assayed sigDE mRNA from cells heat-treated at 60°C, 100°C, and 121°C (autoclaved), and found that mRNA was degraded in autoclaved bacterial samples. These results showed that the sigDE transcript is a suitable mRNA target for rt-RT-PCR with samples pretreated at 121°C. Thus, an rt-RT-PCR using sigDE primers was developed for the detection of viable Salmonella. An RNA internal amplification control was constructed by overlap extension PCR, synthesized using in vitro transcription with a T7 RNA polymerase promoter, and incorporated into the rt-RT-PCR system to eliminate false-negative results. The rt-RT-PCR system has the capability of specifically detecting all the tested S. enterica serovars, and the detection limit of this assay with cultures of Salmonella Typhimurium ATCC 13311 was 10(1) colony-forming units (CFU)/mL. After 18-h enrichment, sigDE-based rt-RT-PCR could detect as low as 10(0) CFU/mL of Salmonella from egg broth and milk.

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“…Recent advancements in molecular diagnostics have shown the potential to reduce assay time to less than 10 min using plasmonics and microfluidic techniques [1][2][3] . However, in the case of most commercialized molecular diagnostics, a sample preparation step is inevitably involved, leading to a relatively lengthy diagnosis time of up to several hours [4][5][6][7] (See Supplementary Tables 1, 2).…”

mentioning

confidence: 99%

Rapid deep learning-assisted predictive diagnostics for point-of-care testing

Lee,

Park,

Woo

et al. 2024

Nat Commun

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Prominent techniques such as real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and rapid kits are currently being explored to both enhance sensitivity and reduce assay time for diagnostic tests. Existing commercial molecular methods typically take several hours, while immunoassays can range from several hours to tens of minutes. Rapid diagnostics are crucial in Point-of-Care Testing (POCT). We propose an approach that integrates a time-series deep learning architecture and AI-based verification, for the enhanced result analysis of lateral flow assays. This approach is applicable to both infectious diseases and non-infectious biomarkers. In blind tests using clinical samples, our method achieved diagnostic times as short as 2 minutes, exceeding the accuracy of human analysis at 15 minutes. Furthermore, our technique significantly reduces assay time to just 1-2 minutes in the POCT setting. This advancement has the potential to greatly enhance POCT diagnostics, enabling both healthcare professionals and non-experts to make rapid, accurate decisions.

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Sample Preparation for PCR

Cited by 9 publications

References 27 publications

Real-time reverse-transcriptase PCR for Salmonella Typhimurium detection from lettuce and tomatoes

Real-time reverse-transcriptase PCR for Salmonella Typhimurium detection from lettuce and tomatoes

Development of asigDE-Based Real-Time Reverse-Transcriptase PCR for the Detection of ViableSalmonella enterica

Rapid deep learning-assisted predictive diagnostics for point-of-care testing

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