Fast on-site diagnosis of influenza A virus by Palm PCR and portable capillary electrophoresis.

A method combining Palm polymerase chain reaction (PCR) and portable capillary electrophoresis (CE) was developed for rapid on-site analysis of influenza A (H1N1) virus. The portable CE system was suitable for rapid diagnosis which was able to detect a sample in ∼4 min after sample loading, while the 'Palm PCR' system allowed for high-speed nucleic acid amplification in ∼16 min. The analysis time from DNA sample to analysis of amplified target DNA molecule was only ∼20 min, which was significantly less than slab gel electrophoresis with other commercially available PCR machine. When the 100-bp DNA ladder was separated, the relative standard deviation values (n=5) for the migration times and peak areas of the 100 and 200-bp DNA molecules were 0.26 and 8.9%. The detection limits were 6.3 and 7.2 pg/μL, respectively. The combined method was also able to identify two influenza A-associated genes (the HA and NP genes of the novel H1N1 influenza). CE separation was achieved with a sieving matrix of 1% poly(vinylpyrrolidone) (Mr=1,300,000) in 1× TBE buffer (pH 8.45). The combined Palm PCR-portable CE system should provide an improved, fast on-site molecular genetic diagnostic method.

[1]  Amy E Herr,et al.  Integrated Microfluidic Platform for Oral Diagnostics , 2007, Annals of the New York Academy of Sciences.

[2]  S. Kang,et al.  Portable capillary electrophoresis system for identification of cattle breeds based on DNA mobility , 2010, Electrophoresis.

[3]  Nick Harris,et al.  Review on the development of truly portable and in-situ capillary electrophoresis systems , 2013 .

[4]  Hee Sung Hwang,et al.  Simultaneous quantitative detection of 12 pathogens using high‐resolution CE‐SSCP , 2010, Electrophoresis.

[5]  M. B. Grace,et al.  Development and assessment of a quantitative reverse transcription-PCR assay for simultaneous measurement of four amplicons. , 2003, Clinical chemistry.

[6]  Yi Guan,et al.  Emergence of a novel swine-origin influenza A virus (S-OIV) H1N1 virus in humans. , 2009, Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology.

[7]  E. Yeung,et al.  Laser fluorescence detector for capillary electrophoresis , 1992 .

[8]  Sangwei Lu,et al.  Detection and subtyping of influenza A virus based on a short oligonucleotide microarray. , 2009, Diagnostic microbiology and infectious disease.

[9]  Quan Cheng,et al.  Detection of influenza virus: traditional approaches and development of biosensors , 2005, Analytical and bioanalytical chemistry.

[10]  R. Rodríguez-Ramirez,et al.  Development of a polymerase chain reaction and capillary gel electrophoresis method for the detection of chicken or turkey meat in heat-treated pork meat mixtures. , 2011, Analytica chimica acta.

[11]  Dermot Brabazon,et al.  Portable capillary-based (non-chip) capillary electrophoresis , 2010 .

[12]  Hadamard transform capillary electrophoresis combined with laser-induced fluorometry using electrokinetic injection. , 2006, Analytica chimica acta.

[13]  Kevin W Plaxco,et al.  Rapid, sequence-specific detection of unpurified PCR amplicons via a reusable, electrochemical sensor. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[14]  G. Kochs,et al.  Singleplex real-time RT-PCR for detection of influenza A virus and simultaneous differentiation of A/H1N1v and evaluation of the RealStar influenza kit , 2010, Journal of Clinical Virology.

[15]  Rosanne M Guijt,et al.  Identification of inorganic improvised explosive devices by analysis of postblast residues using portable capillary electrophoresis instrumentation and indirect photometric detection with a light-emitting diode. , 2007, Analytical chemistry.

[16]  Miss A.O. Penney.  (b) , 1974, The New Yale Book of Quotations.

[17]  Hee Sung Hwang,et al.  Recent developments in CE‐based detection methods for food‐borne pathogens , 2010, Electrophoresis.

[18]  Priti Thakur,et al.  Expression, purification, and characterisation of nesiritide using an E. coli expression system , 2011 .

[19]  M. Reijans,et al.  Evaluation of MeningoFinder, a Novel Multiplex Ligation-Dependent Probe Amplification Assay for Simultaneous Detection of Six Virus Species Causing Central Nervous System Infections , 2009, Journal of Clinical Microbiology.

[20]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[21]  P. Patel,et al.  rapidSTRIPE H1N1 Test for Detection of the Pandemic Swine Origin Influenza A (H1N1) Virus , 2011, Journal of Clinical Microbiology.

[22]  S. Lunte,et al.  Microchip capillary electrophoresis/ electrochemistry , 2001, Electrophoresis.

[23]  P E Klapper,et al.  Multiplex PCR: Optimization and Application in Diagnostic Virology , 2000, Clinical Microbiology Reviews.

[24]  M. Gramer,et al.  Characterization of Influenza A Outbreaks in Minnesota Swine Herds and Measures Taken to Reduce the Risk of Zoonotic Transmission , 2012, Zoonoses and public health.

[25]  J. Derisi,et al.  Microarray-based detection and genotyping of viral pathogens , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Sam F. Y. Li,et al.  Rapid analysis of native neomycin components on a portable capillary electrophoresis system with potential gradient detection , 2006, Analytical and bioanalytical chemistry.