A next-generation gas chromatograph-molecular rotational resonance (MRR) spectrometer (GC-MRR) with instrumental improvements and higher sensitivity is described. MRR serves as a structural information-rich detector for GC with extremely narrow linewidths and capabilities surpassing 1 H nuclear magnetic resonance/Fourier transform infrared spectroscopy/mass spectrometry (MS) while offering unparalleled specificity in regard to a molecule's three-dimensional structure. With a Fabry−Peŕot cavity and a supersonic jet incorporated into a GC-MRR, dramatic improvements in sensitivity for molecules up to 244 Da were achieved in the microwave region compared to the only prior work, which demonstrated the GC-MRR idea for the first time with millimeter waves. The supersonic jet cools the analytes to ∼2 K, resulting in a limited number of molecular rotational and vibrational levels and enabling us to obtain stronger GC-MRR signals. This has allowed the limits of detection of the GC-MRR to be comparable to a GC thermal conductivity detector with an optimized choice of gases. The performance of this GC-MRR system is reported for a range of molecules with permanent dipole moments, including alcohols, nitrogen heterocyclics, halogenated compounds, dioxins, and nitro compounds in the molecular mass range of 46−244 Da. The lowest amount of any substance yet detected by MRR in terms of mass is reported in this work. A theoretically unexpected finding is reported for the first time about the effect of the GC carrier gas (He, Ne, and N 2 ) on the sensitivity of the analysis in the presence of the gas driving the supersonic jet (He, Ne, and N 2 ) in the GC-MRR. Finally, the idea of total molecule monitoring in the GC-MRR analogous to selected ion monitoring in GC-MS is illustrated. Structural isomers and isotopologues of bromobutanes and bromonitrobenzenes are used to demonstrate this concept.
Verticillium wilt, which is caused by the fungus Verticillium dahliae, is one of the most important olive diseases worldwide. There are many ways to extract DNA from plant pathogenic fungi and from plant tissues for molecular-based diagnostic assays. LAMP is a new and sensitive molecular-based technique used for detection of plant pathogenic agents with minimum requirements needed. In this study, we tried to achieve a simple, cost effective and efficient method of DNA extraction from both Verticillium dahliae fungus and from infected wood samples in order to run a loop-mediated isothermal amplification (LAMP) assay. Efficiency of three DNA isolation methods from both mycelia and infected wood samples was evaluated. For this purpose, wood samples from infected olive trees were collected from Tarom region in Zanjan province and the samples were cultured on the media. The fungus was isolated and identified as V. dahliae based on morphological features. Then the genomic DNA was extracted using traditional CTAB method, fast NaOH method and direct isolation method from infected wood samples. After assessment of the quality and the quantity of the extracted DNA samples, a LAMP assay was ran using specific primer pairs and the DNA templates extracted using three different methods. In spite of the significant differences in the quantity of DNA samples, LAMP assay could successfully detect the fungus in all samples. The improved direct isolation of the DNA of V. dahlia from infected wood, followed by a LAMP assay could considerably shortened the detection process of the fungus and hence is a suitable method for screening of olive trees and saplings against Verticillium wilt disease.
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