Rapid pathogen detection using a microchip PCR array instrument

Abstract

An array of PCR microchips for rapid, parallel testing of samples for pathogenic microbes is described. The instrument, called the Advanced Nucleic Acid Analyzer (ANAA), utilizes 10 silicon reaction chambers with thin-film resistive heaters and solid-state optics. Features of the system include efficient heating and real-time monitoring, low power requirements for battery operation, and no moving parts for reliability and ruggedness. We analyzed cultures of Erwinia herbicola vegetative cells, Bacillus subtilis spores, and MS2 virions, which simulated pathogenic microbes such as Yersinia pestis, Bacillus anthracis spores, and Venezuelan equine encephalitis, respectively. Detection of microbes was achieved in as little as 16 min with detection limits of 105–107 organisms/L (102–104 organisms/mL).

Physical Collection Efficiency of Filter Materials for Bacteria and Viruses

“…In addition, MS2 bacteriophages have previously been used a surrogate for poliovirus and many other enteric viruses due to similarity of their characteristics. Several investigators utilized MS2 as a simulant of pathogenic viral strains (Belgrader et al, 1998;Alvarez et al, 2000;Shin and Sobsey, 2003;Thomas et al, 2004;Tseng and Li, 2005b). The MS2 bacteriophage stock solution was prepared from a freeze-dried phage stock vial (ATCC 15597-B1) by adding 9 ml of Luria-Bertani broth, which had been made using ultra-filtered deionized water.…”

Gold nanoparticle-assisted polymerase chain reaction: effects of surface ligands, nanoparticle shape and material

“…4 As a result of the poor purication of template DNA, primer-template mismatches, spontaneous formation of primer dimers, and non-optimized PCRmixture ratio and thermocycling conditions, the formation of the target amplicon may be accompanied by non-specic side products called PCR artifacts. To x this problem, several enhancers have been introduced: (1) various additives in PCR mixture, such as betaine, 5 dithiothreitol, 6 dimethyl sulfoxide, 7 single-stranded DNA-binding protein (SSB); 8 (2) instrumental design, including development of thermocyclers with precise heating/cooling rates; 9,10 (3) optimization of PCR system through optimal Mg 2+ concentration, cycle numbers and proper primer design; [11][12][13] (4) enzyme modication; 14,15 and (5) new touchdown 16 and nested 17 PCR strategies. Although these tools improve the PCR outcome, yet they are not all-purpose and the optimization protocol can be case dependent.…”

Multiplex PCR for detection of Enterobacteriaceae in blood

“…The time required to complete the assay was only 3 hours. Recent development of thermocyclers and spectrofluorometers that use silicon chips has reduced the size of the instrumentation needed and permitted assays to be completed even faster—in only a few minutes 31,32 . Thus, the assay provides both the speed and extreme sensitivity that might be suitable for detecting the small numbers of bacteria thought to contaminate blood during the initial days of storage.…”