Genome sequencing in microfabricated high-density picolitre reactors.

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

Rapid identification of dengue virus by reverse transcription-polymerase chain reaction using field-deployable instrumentation.

Dengue virus universal and dengue serotype 1 to 4, fluorogenic probe hydrolysis (TaqMan), reverse transcription-polymerase chain reaction assays were developed for screening and serotype identification of infected mosquito vectors and human sera using a field-deployable, fluorometric thermocycler. Dengue universal and dengue 1 to 4 serotype assay in vitro sensitivity and specificity results were 100% concordant when tested with total nucleic acid extracts of multiple strains of dengue serotype 1 to 4, yellow fever, Japanese encephalitis, West Nile, and St. Louis encephalitis viruses. The in vitro sensitivity and specificity results for all five assays were concordant when tested with a blind panel of 27 dengue virus-infected mosquitoes, 21 non-dengue (yellow fever, West Nile, or St. Louis encephalitis) flavivirus-infected mosquitoes, and 11 uninfected mosquitoes and with clinical specimens consisting of a human serum panel of eight dengue viremic and 31 non-dengue-infected febrile patient serum samples. No cross-reaction occurred with vector species or human genomic DNA. Sample processing and polymerase chain reaction required <2 hours.

A rapid, single-step multiplex reverse transcription-PCR assay for the detection of human H1N1, H3N2, and B influenza viruses.

BACKGROUND: Influenza is a viral respiratory pathogen responsible for frequent seasonal epidemics. There are currently three major human influenza viruses in global circulation, H1N1, H3N2 and B. OBJECTIVES: A one-step multiplex reverse transcription (RT)-polymerase chain reaction (PCR) assay targeting the HA1 segment of the human hemagglutinin gene was developed as a rapid surveillance method. STUDY DESIGN: A researcher-blind study was performed using 112 randomly selected, culture-positive clinical samples collected through the Department of Defense (Global Emerging Infectious Surveillance (DOD-GEIS) influenza network during the 2000-2001 influenza season. Three subtype specific primer sets capable of producing PCR products with base-pair lengths of 585, 402 and 290 corresponding to influenza H1, H3, and B subtypes, respectively, were utilized together in a one step, one tube, reaction. RESULTS: Multiplex primers were able to simultaneously type, and subtype 100% (112/112) of positive cultures. CONCLUSIONS: The results confirm that this assay is a highly sensitive and timely surveillance tool for rapid detection and simultaneous subtyping of clinical influenza specimens isolated worldwide.

A real-time fluorescence polymerase chain reaction assay for the identification of Yersinia pestis using a field-deployable thermocycler.

Real-time fluorescence polymerase chain reaction is a microbial identification method that can provide rapid and accurate results using a field-deployable thermocycler, the RAPID ("ruggedized" advanced pathogen identification device). A Yersinia pestis-specific TaqMan assay required approximately 75 minutes and achieved a sensitivity of 100 fg of Y. pestis genomic DNA (20 genome equivalents). Specificity testing against a genomic DNA cross-reaction panel comprised of 22 bacterial species encountered in the respiratory tract resulted in no false positives. No cross-reaction occurred with human genomic DNA.

Genetic and antigenic analysis of the first A/New Caledonia/20/99-like H1N1 influenza isolates reported in the Americas.

From February through May of 1999, 13 cases of Influenza A virus (FLUAV), type H1N1 were reported at a Department of Defense influenza surveillance sentinel site in Lima, Peru. Genetic and antigenic analysis by hemagglutination inhibition and direct nucleotide sequencing of the HA1 region of the hemagglutinin gene were performed on two isolates, A/Peru/1641/99 and A/Peru/1798/99. Both isolates were distinct from the Bayern/7/95-like viruses circulating in the Americas and closely related to a Beijing/262/95-like variant, A/New Caledonia/20/99. With the exception of travel-related cases, the detection of these isolates represents the first appearance of New Caledonia/20/99-like viruses in the Americas. Since the characterization of these Peru isolates, a number of New Caledonia/20/99-like viruses have been reported worldwide. For the 2000/01 and 2001/02 influenza seasons, the World Health Organization (WHO) has recommended the inclusion of A/New Caledonia/20/99 as the H1N1 vaccine component for both the southern and northern hemispheres.

Real-time PCR detection of salmonella in suspect foods from a gastroenteritis outbreak in kerr county, Texas.

In June 2001, an outbreak of acute gastroenteritis among 109 attendees of a church picnic in Kerr County, Texas, was reported. A 5'-nuclease PCR assay was used to screen for Salmonella in nine food items from the buffet line. Barbeque chicken B tested positive for Salmonella, and no amplification was detected in the remaining food items. These PCR findings were consistent with culture results and were confirmed by direct nucleotide sequencing. Salmonella enterica serotype Panama was cultured from both food and patient stool samples.

A massively parallel PicoTiterPlate based platform for discrete picoliter-scale polymerase chain reactions.

We demonstrate successful, simultaneous polymerase chain reaction (PCR) amplification of up to 300 000 discrete reactions in a novel platform, the PicoTiterPlate. In addition to elevated throughput, the PicoTiterPlate based amplifications (PTPCR) can be performed in extremely small volumes: individual reactions volumes are as low as 39.5 pL, with a total 15.3 microL reaction volume for the entire PicoTiterPlate. The bulk PTPCR product can be recovered and assayed with real-time PCR, or discrete PTPCR products can be driven to solid supports, enabling downstream applications such as translation/transcription or sequencing.