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Highly sensitive identification of foodborne pathogenic Listeria monocytogenes using single-phase continuous-flow nested PCR microfluidics with on-line fluorescence detection

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Abstract

Highly sensitive detection of foodborne pathogens such as Listeria monocytogenes (L. monocytogenes) is crucial to the prevention and recognition of problems related to public health and legal repercussions, due to “zero tolerance” standard adopted for food safety in many countries. Here we first propose a single-phase continuous-flow nested polymerase chain reaction (SP-CF-NPCR) strategy for identification of the low level of L. monocytogenes on an integrated microfluidic platform. The PCR reactor is constructed by a disposable capillary embedded in the grooved heating column, coupled with a fluorescence microscopy for on-line semi-quantitative end-point fluorescence detection. As a proof-of-concept microfluidic system, the nested PCR is performed in a continuous-flow format without the need of any non-aqueous oil or solvent. On this device, the performance of nested PCR amplification has been evaluated by investigating the effect of reaction parameters, including polymerase concentration, flow rates, and template DNA concentration. In addition, the types of samples the presented system can accept, such as the unpurified DNA samples and artificially contaminated clinical stool samples were also evaluated. With the optimized reaction parameters, 0.2 copies/μL of genomic DNA from L. monocytogenes can be detected on the presented device. To our knowledge, this is the highest detection sensitivity in single-phase continuous-flow PCR microsystems reported so far. The high sensitivity of the analysis method, combined with the flexibility of reaction volumes and convenience of continuous operation, renders it to be further developed for potential analytical and diagnostic applications.

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References

  • Anderson RC, Su X, Bogdan GJ, Fenton J (2000) A miniature integrated device for automated multistep genetic assays. Nucleic Acids Res 28(12):e60

    Article  Google Scholar 

  • Batt CA (2007) Materials science—food pathogen detection. Science 316(5831):1579–1580

    Article  Google Scholar 

  • Beyor N, Yi L, Seo TS, Mathies RA (2009) Integrated capture, concentration, polymerase chain reaction, and capillary electrophoretic analysis of pathogens on a chip. Anal Chem 81(9):3523–3528

    Article  Google Scholar 

  • Cady NC, Stelick S, Kunnavakkam MV, Batt CA (2005) Real-time PCR detection of Listeria monocytogenes using an integrated microfluidics platform. Sens Actuat B 107(1):332–341

    Article  Google Scholar 

  • Chen ZY, Qian SZ, Abrams WR, Malamud D, Bau HH (2004) Thermosiphon-based PCR reactor: experiment and modeling. Anal Chem 76(13):3707–3715

    Article  Google Scholar 

  • Chen JF, Wabuyele M, Chen HW, Patterson D, Hupert M, Shadpour H, Nikitopoulos D, Soper SA (2005a) Electrokinetically synchronized polymerase chain reaction microchip fabricated in polycarbonate. Anal Chem 77(2):658–666

    Article  Google Scholar 

  • Chen ZY, Wang J, Qian SZ, Bau HH (2005b) Thermally-actuated, phase change flow control for microfluidic systems. Lab Chip 5(11):1277–1285

    Article  Google Scholar 

  • Chen L, West J, Auroux PA, Manz A, Day PJR (2007) Ultrasensitive PCR and real-time detection from human genomic samples using a bidirectional flow microreactor. Anal Chem 79(23):9185–9190

    Article  Google Scholar 

  • Crabtree HJ, Lauzon J, Morrissey YC, Taylor BJ, Liang T, Johnstone RW, Stickel AJ, Manage DP, Atrazhev A, Backhouse CJ, Pilarski LM (2012) Inhibition of on-chip PCR using PDMS-glass hybrid microfluidic chips. Microfluid Nanofluid 13(3):383–398

    Article  Google Scholar 

  • Crews N, Wittwer C, Gale B (2008) Continuous-flow thermal gradient PCR. Biomed Microdevices 10(2):187–195

    Article  Google Scholar 

  • Hashimoto M, Chen PC, Mitchell MW, Nikitopoulos DE, Soper SA, Murphy MC (2004) Rapid PCR in a continuous flow device. Lab Chip 4(6):638–645

    Article  Google Scholar 

  • Hua Z, Rouse JL, Eckhardt AE, Srinivasan V, Pamula VK, Schell WA, Benton JL, Mitchell TG, Pollack MG (2010) Multiplexed real-time polymerase chain reaction on a digital microfluidic platform. Anal Chem 82(6):2310–2316

    Article  Google Scholar 

  • Kopp MU, de Mello AJ, Manz A (1998) Chemicalf amplification: continuous-flow PCR on a chip. Science 280(5366):1046–1048

    Article  Google Scholar 

  • Li YY, Zhang CS, Xing D (2011a) Integrated microfluidic reverse transcription-polymerase chain reaction for rapid detection of food- or waterborne pathogenic rotavirus. Anal Biochem 415(2):87–96

    Article  Google Scholar 

  • Li YY, Zhang CS, Xing D (2011b) Fast identification of foodborne pathogenic viruses using continuous-flow reverse transcription-PCR with fluorescence detection. Microfluid Nanofluid 10(2):367–380

    Article  Google Scholar 

  • Liu RH, Yang JN, Lenigk R, Bonanno J, Grodzinski P (2004) Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection. Anal Chem 76(7):1824–1831

    Article  Google Scholar 

  • Liu HB, Ramalingam N, Jiang Y, Dai CC, Hui KM, Gong HQ (2009) Rapid distribution of a liquid column into a matrix of nanoliter wells for parallel real-time quantitative PCR. Sens Actuat B 135(2):671–677

    Article  Google Scholar 

  • Lok KS, Lee PPF, Kwok YC, Nam-Trung N (2012) Nested PCR in magnetically actuated circular closed-loop PCR microchip system. Microchim Acta 177(1–2):111–117

    Google Scholar 

  • Manage DP, Morrissey YC, Stickel AJ, Lauzon J, Atrazhev A, Acker JP, Pilarski LM (2011) On-chip PCR amplification of genomic and viral templates in unprocessed whole blood. Microfluid Nanofluid 10(3):697–702

    Article  Google Scholar 

  • Nakayama T, Kurosawa Y, Furui S, Kerman K, Kobayashi M, Rao SR, Yonezawa Y, Nakano K, Hino A, Yamamura S, Takamura Y, Tamiya E (2006) Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications. Anal Bioanal Chem 386(5):1327–1333

    Article  Google Scholar 

  • Njoroge SK, Witek MA, Battle KN, Immethun VE, Hupert ML, Soper SA (2011) Integrated continuous flow polymerase chain reaction and micro-capillary electrophoresis system with bioaffinity preconcentration. Electrophoresis 32(22):3221–3232

    Article  Google Scholar 

  • Northrup MA, Ching MT, White RM, Watson RT (1993) DNA amplification in a microfabricated reaction chamber. In: Proceeding of the 7th international conference on solid state sensors and actuators, Yokohama, Japan, pp 924–926

  • Park N, Kim S, Hahn JH (2003) Cylindrical compact thermal-cycling device for continuous-flow polymerase chain reaction. Anal Chem 75(21):6029–6033

    Article  Google Scholar 

  • Park S, Zhang Y, Lin S, Wang TH, Yang S (2011) Advances in microfluidic PCR for point-of-care infectious disease diagnostics. Biotechnol Adv 29(6):830–839

    Article  Google Scholar 

  • Peham JR, Grienauer W, Steiner H, Heer R, Vellekoop MJ, Noehammer C, Wiesinger-Mayr H (2011) Long target droplet polymerase chain reaction with a microfluidic device for high-throughput detection of pathogenic bacteria at clinical sensitivity. Biomed Microdevices 13(3):463–473

    Article  Google Scholar 

  • Pjescic I, Crews N (2012) Genotyping from saliva with a one-step microdevice. Lab Chip 12(14):2514–2519

    Article  Google Scholar 

  • Polini A, Mele E, Sciancalepore AG, Girardo S, Biasco A, Camposeo A, Cingolani R, Weitz DA, Pisignano D (2010) Reduction of water evaporation in polymerase chain reaction microfluidic devices based on oscillating-flow. Biomicrofluidics 4(3):036502

    Article  Google Scholar 

  • Prakash AR, Amrein M, Kaler KVIS (2008) Characteristics and impact of Taq enzyme adsorption on surfaces in microfluidic devices. Microfluid Nanofluid 4(4):295–305

    Article  Google Scholar 

  • Qiu X, Mauk MG, Chen D, Liu C, Bau HH (2010) A large volume, portable, real-time PCR reactor. Lab Chip 10(22):3170–3177

    Article  Google Scholar 

  • Qiu X, Chen D, Liu C, Mauk MG, Kientz T, Bau HH (2011) A portable, integrated analyzer for microfluidic-based molecular analysis. Biomed Microdevices 13(5):809–817

    Article  Google Scholar 

  • Ramalingam N, Liu HB, Dai CC, Jiang Y, Wang H, Wang Q, Hui MK, Gong HQ (2009) Real-time PCR array chip with capillary-driven sample loading and reactor sealing for point-of-care applications. Biomed Microdevices 11(5):1007–1020

    Article  Google Scholar 

  • Ramalingam N, Rui Z, Liu HB, Dai CC, Kaushik R, Ratnaharika B, Gong HQ (2010) Real-time PCR-based microfluidic array chip for simultaneous detection of multiple waterborne pathogens. Sens Actuat B 145(1):543–552

    Article  Google Scholar 

  • Ritzi-Lehnert M, Himmelreich R, Attig H, Claussen J, Dahlke R, Grosshauser G, Holzer E, Jeziorski M, Schaeffer E, Wende A, Werner S, Wiborg JO, Wick I, Drese KS, Rothmann T (2011) On-chip analysis of respiratory viruses from nasopharyngeal samples. Biomed Microdevices 13(5):819–827

    Article  Google Scholar 

  • Schaerli Y, Hollfelder F (2009) The potential of microfluidic water-in-oil droplets in experimental biology. Mol BioSyst 5(12):1392–1404

    Article  Google Scholar 

  • Sciancalepore AG, Polini A, Mele E, Girardo S, Cingolani R, Pisignano D (2011) Rapid nested-PCR for tyrosinase gene detection on chip. Biosens Bioelectron 26(5):2711–2715

    Article  Google Scholar 

  • Shi X, Lin LI, Chen SY, Chao SH, Zhang WW, Meldrum DR (2011) Real-time PCR of single bacterial cells on an array of adhering droplets. Lab Chip 11(13):2276–2281

    Article  Google Scholar 

  • Sun Y, Dhumpa R, Bang DD, Hogberg J, Handberg K, Wolff A (2011a) A lab-on-a-chip device for rapid identification of avian influenza viral RNA by solid-phase PCR. Lab Chip 11(8):1457–1463

    Article  Google Scholar 

  • Sun Y, Dhumpa R, Bang DD, Handberg K, Wolff A (2011b) DNA microarray-based solid-phase RT-PCR for rapid detection and identification of influenza virus type A and subtypes H5 and H7. Diagn Microbiol Infect Dis 69(4):432–439

    Article  Google Scholar 

  • Wang F, Burns MA (2009) Performance of nanoliter-sized droplet-based microfluidic PCR. Biomed Microdevices 11(5):1071–1080

    Article  Google Scholar 

  • Wang F, Burns MA (2010) Droplet-based microsystem for multi-step bioreactions. Biomed Microdevices 12(3):533–541

    Article  Google Scholar 

  • Yang JN, Liu YJ, Rauch CB, Stevens RL, Liu RH, Lenigk R, Grodzinski P (2002) High sensitivity PCR assay in plastic micro reactors. Lab Chip 2(4):179–187

    Article  Google Scholar 

  • Zeng Y, Novak R, Shuga J, Smith MT, Mathies RA (2010) High-performance single cell genetic analysis using microfluidic emulsion generator arrays. Anal Chem 82(8):3183–3190

    Article  Google Scholar 

  • Zhang CS, Xing D (2007) Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends. Nucleic Acids Res 35(13):4223–4237

    Article  Google Scholar 

  • Zhang CS, Xing D (2009) Parallel DNA amplification by convective polymerase chain reaction with various annealing temperatures on a thermal gradient device. Anal Biochem 387(1):102–112

    Article  Google Scholar 

  • Zhang CS, Xing D (2010) Single-molecule DNA amplification and analysis using microfluidics. Chem Rev 110(8):4910–4947

    Article  Google Scholar 

Download references

Acknowledgments

This research is supported by the National Natural Science Foundation of China (61072030), the National Basic Research Program of China (2010CB732602), the Key Program of NSFC-Guangdong Joint Funds of China (U0931005), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT0829).

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Authors and Affiliations

  1. MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, 510631, China

    Bowen Shu, Chunsun Zhang & Da Xing

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  1. Bowen Shu
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  2. Chunsun Zhang
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  3. Da Xing
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Correspondence to Chunsun Zhang or Da Xing.

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Shu, B., Zhang, C. & Xing, D. Highly sensitive identification of foodborne pathogenic Listeria monocytogenes using single-phase continuous-flow nested PCR microfluidics with on-line fluorescence detection. Microfluid Nanofluid 15, 161–172 (2013). https://doi.org/10.1007/s10404-013-1138-4

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