GC/MS Method for Positive Detection of Bacillus anthracis Endospores

Abstract

A simple method was developed for detection of Bacillus anthracis (BA) endospores and for differentiation of them from other species in the Bacillus cereus group. Chemical profiles that include lipids (i.e., fatty acids), carbohydrates (i.e., sugars), and the spore-specific biomarker, dipicolinic acid, were generated by one-step thermochemolysis (TCM) at 140 °C in 5 min to provide specific biomarker signatures. Anthrose, which is a biomarker characteristic of the B. cereus group of bacteria, was determined from a fragment produced by TCM. Surprisingly, several virulent BA strains contained very low levels of anthrose, which confounded their detection. A statistical discrimination algorithm was constructed using a combination of biomarkers, which was robust against different growth conditions (medium and temperature). Fifteen endospore-forming Bacillus species were confirmed in a statistically designed test (~90%) using the algorithm, including six BA strains (four virulent isolates), five B. thuringiensis (BT) isolates, and one isolate each for B. cereus (BC), B. mycoides (BM), B. atrophaeus (BG), and B. subtilis (BS). The detection limit for B. anthracis was found to be 50,000 endospores, on the basis of the GC/MS detection limits for 3-methyl-2-butenoic acid methyl ester, which is the biomarker derived from TCM of anthrose.

Gas chromatography-mass spectrometry method for rapid identification and differentiation ofBurkholderia pseudomalleiandBurkholderia malleifrom each other,Burkholderia thailandensisand several members of theBurkholderia cepaciacomplex

“…The SPME fibre was inserted through the rubber septum of the capped vial (Li et al . ). Roughly, half of the fibre was submerged in the liquid phase, and half was exposed to the vapour phase.…”

“…The TCM procedure was a modified version of the protocol that was used to generate biomarkers from the spores of several Bacillus species (Li et al . ). A statistical discrimination algorithm was then constructed using a combination of biomarkers, and the identities of the different species of Burkholderia were confirmed in a statistically designed test using the algorithm.…”

Detection of Bacillus anthracis spores by super-paramagnetic lateral-flow immunoassays based on “Road Closure”

“…2 and 3), indicating that as few as 500-700 B. anthracis spores can be recognized. This is not only far below the estimated 50% lethal dose (LD 50 ) for humans of 3000-50,000 spores (Eubanks et al, 2007), but is also more sensitive than many other immunological detection methods which are combined with advanced instrumentation (Li et al, 2012;Wang et al, 2009a). Similar results can be achieved for low concentrations of powdery substances including 5-15% milk and 5% starch (data not shown).…”

“…In addition to conventional bacteriological assays, various techniques are capable of identifying B. anthracis, including immunoassays (Morel et al, 2012), DNA sequencing , biosensor detection systems (Hao et al, 2009;Wang et al, 2009a), Raman microspectroscopy (Arora et al, 2012), Gas chromatography-Mass spectrometry (Li et al, 2012) and other advanced approaches based on nanomaterials . These methods tend to have long detection time, require the use of expensive devices, complicated protocols or experienced operators.…”

Using color histograms and SPA-LDA to classify bacteria

“…They include gas chromatography [7,8], capillary electrophoresis [9], mass spectroscopy [6,10], Raman spectroscopy [11], infrared spectroscopy [12][13][14], fluorescence [15,16], and nuclear magnetic resonance [17]. Despite the ability of these instrumental analytical techniques to obtain reliable and accurate results in the identification and classification of microorganisms, the use of digital images presents some advantages, since it has low-cost equipment, avoids the manipulation of the Petri plate where microorganisms are grown, and does not require the knowledge of a skillful microbiologist.…”