Quantitative or digital PCR? A comparative analysis for choosing the optimal one for biosensing applications

Zhang, Haoqing; Cao, Lei; Brodsky, Jan; Gablech, Imrich; Xu, Feng; Li, Zedong; Korabecna, Marie; Neuzil, Pavel

doi:10.1016/j.trac.2024.117676

Cited by 4 publications

(3 citation statements)

References 139 publications

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“…In response, culture-independent methods based on DNA, such as simultaneous real-time quantitative PCR (multiplex RT qPCR) or conventional real-time quantitative PCR (RT qPCR), have been reported as Microorganisms 2024, 12, 1223 2 of 17 promising methods because they allow the specific identification and quantification of microorganisms within a few hours [16][17][18], such as proposed RT qPCR assays by the U.S. Environmental Protection Agency for FIB detection [19]. Additionally, multiplex RT qPCR reduces the required sample volume, cost, and handling of assays compared to RT qPCR or other independent-culture methods [20][21][22].…”

Section: Introductionmentioning

confidence: 99%

Development of Multiplex RT qPCR Assays for Simultaneous Detection and Quantification of Faecal Indicator Bacteria in Bathing Recreational Waters

Carrasco-Acosta,

Garcia-Jimenez

2024

Microorganisms

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In this study, we designed and validated in silico and experimentally a rapid, sensitive, and specific multiplex RT qPCR for the detection and quantification of faecal indicator bacteria (FIB) used as microbiological references in marine bathing water regulations (Escherichia coli and intestinal enterococci). The 16S rRNA gene was used to quantify group-specific enterococci and Escherichia/Shigella and species-specific such as Enterococcus faecalis and E. faecium. Additionally, a ybbW gene encoding allantoin transporter protein was used to detect E. coli. An assessment of marine coastal systems (i.e., marine water and sediment) revealed that intestinal enterococci were the predominant group compared to Escherichia/Shigella. The low contribution of E. faecalis to the intestinal enterococci group was reported. As E. faecalis and E. faecium were reported at low concentrations, it is assumed that other enterococci of faecal origin are contributing to the high gene copy number of this group-specific enterococci. Moreover, low 16S rRNA gene copy numbers with respect to E. faecalis and E. faecium were reported in seawater compared to marine sediment. We conclude that marine sediments can affect the quantification of FIBs included in bathing water regulations. Valuing the quality of the marine coastal system through sediment monitoring is recommended.

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Section: Introductionmentioning

confidence: 99%

Development of Multiplex RT qPCR Assays for Simultaneous Detection and Quantification of Faecal Indicator Bacteria in Bathing Recreational Waters

Carrasco-Acosta,

Garcia-Jimenez

2024

Microorganisms

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“…3,4 Digital detection provides precise measurements and allows for accurate quantification of small amounts of targets. 5,6 Droplet microfluidics is advantageous for digital detection assays as it offers the capability of generating a large number of droplets for highly efficient sample compartmentalization. 7,8 Such assays are suited for various targets including nucleic acid, protein, and cell, making it extensively employed in biomedical research and clinical diagnostics.…”

mentioning

confidence: 99%

“…A digital detection assay is a quantitative analysis method that relies on large-scale dispersion systems and employs the Poisson distribution algorithm. , This approach estimates the concentration of the detection target by evaluating the proportion of statistically positive units, leading to significant enhancements in detection sensitivity and quantitation accuracy. , Digital detection provides precise measurements and allows for accurate quantification of small amounts of targets. , Droplet microfluidics is advantageous for digital detection assays as it offers the capability of generating a large number of droplets for highly efficient sample compartmentalization. , Such assays are suited for various targets including nucleic acid, protein, and cell, making it extensively employed in biomedical research and clinical diagnostics. − …”

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confidence: 99%

Rapid Detection of Uropathogens Using an Integrated Multiplex Digital Nucleic Acid Detection Assay Powered by a Digital-to-Droplet Microfluidic Device

Xie,

Chen,

Cai

et al. 2024

Anal. Chem.

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The digital nucleic acid detection assay features the capability of absolute quantitation without the need for calibration, thereby facilitating the rapid identification of pathogens. Although several integrated digital nucleic acid detection techniques have been developed, there are still constraints in terms of automation and analysis throughput. To tackle these challenges, this study presents a digital-to-droplet microfluidic device comprising a digital microfluidics (DMF) module at the bottom and a droplet microfluidics module at the top. Following sample introduction, the extraction of nucleic acid and the dispensation of nucleic acid elution for mixing with the multiple amplification reagents are carried out in the DMF module. Subsequently, the reaction droplets are transported to the sample inlet of the droplet microfluidic module via a liquid outlet, and then droplet generation in four parallel units within the droplet microfluidics module is actuated by negative pressure generated by a syringe vacuum. The digital-to-droplet microfluidic device was employed to execute an integrated multiplex digital droplet nucleic acid detection assay (imDDNA) incorporating loop-mediated isothermal amplification (LAMP). This assay was specifically designed to enable simultaneous detection of four uropathogens, namely, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterococcus faecalis. The entire process of the imDDNA is completed within 75 min, with a detection range spanning 5 orders of magnitude (9.43 × 10−2.86 × 10 4 copies μL −1 ). The imDDNA was employed for the detection of batched clinical specimens, showing a consistency of 91.1% when compared with that of the conventional method. The imDDNA exhibits simplicity in operation and accuracy in quantification, thus offering potential advantages in achieving rapid pathogen detection.

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Early diagnosis and rapid detection of TP53 mutation by REAL-TIME PCR in oral cancer

Nair,

Kannan,

Sivaperumal

2024

Oral Oncology Reports

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Quantitative or digital PCR? A comparative analysis for choosing the optimal one for biosensing applications

Cited by 4 publications

References 139 publications

Development of Multiplex RT qPCR Assays for Simultaneous Detection and Quantification of Faecal Indicator Bacteria in Bathing Recreational Waters

Development of Multiplex RT qPCR Assays for Simultaneous Detection and Quantification of Faecal Indicator Bacteria in Bathing Recreational Waters

Rapid Detection of Uropathogens Using an Integrated Multiplex Digital Nucleic Acid Detection Assay Powered by a Digital-to-Droplet Microfluidic Device

Early diagnosis and rapid detection of TP53 mutation by REAL-TIME PCR in oral cancer

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