Genome-inspired molecular identification in organic matter via Raman spectroscopy

Rapid, non-destructive characterization of molecular level chemistry for organic matter (OM) is experimentally challenging. Raman spectroscopy is one of the most widely used techniques for non-destructive chemical characterization, although it currently does not provide detailed identification of molecular components in OM, due to the combination of diffraction-limited spatial resolution and poor applicability of peak-fitting algorithms. Here, we develop a genome-inspired collective molecular structure fingerprinting approach, which utilizes ab initio calculations and data mining techniques to extract molecular level chemistry from the Raman spectra of OM. We illustrate the power of such an approach by identifying representative molecular fingerprints in OM, for which the molecular chemistry is to date inaccessible using non-destructive characterization techniques. Chemical properties such as aromatic cluster size distribution and H/C ratio can now be quantified directly using the identified molecular fingerprints. Our approach will enable non-destructive identification of chemical signatures with their correlation to the preservation of biosignatures in OM, accurate detection and quantification of environmental contamination, as well as objective assessment of OM with respect to their chemical contents

Theory and Application of Dissociative Electron Capture in Molecular Identification

The coupling of an electron monochromator (EM) to a mass spectrometer (MS) has created a new analytical technique, EM-MS, for the investigation of electrophilic compounds. This method provides a powerful tool for molecular identification of compounds contained in complex matrices, such as environmental samples. EM-MS expands the application and selectivity of traditional MS through the inclusion of a new dimension in the space of molecular characteristics--the electron resonance energy spectrum. However, before this tool can realize its full potential, it will be necessary to create a library of resonance energy scans from standards of the molecules for which EM-MS offers a practical means of detection. Here, an approach supplementing direct measurement with chemical inference and quantum scattering theory is presented to demonstrate the feasibility of directly calculating resonance energy spectra. This approach makes use of the symmetry of the transition-matrix element of the captured electron to discriminate between the spectra of isomers. As a way of validating this approach, the resonance values for twenty-five nitrated aromatic compounds were measured along with their relative abundance. Subsequently, the spectra for the isomers of nitrotoluene were shown to be consistent with the symmetry-based model. The initial success of this treatment suggests that it might be possible to predict negative ion resonances and thus create a library of EM-MS standards.Comment: 18 pages, 7 figure

Strangles: The Molecular Identification and Epidemiology of Streptococcus equi subsp. equi

A conventional PCR diagnostic test was established to confirm the microbiological isolation of Streptococcus equi subsp. equi (S. equi), the causative agent of strangles. This test was based on the amplification of the seeI gene, which is species-specific for S. equi. Further, a multiplex PCR was developed using species-specific primers; to identify the presence of S. equi and two other streptococci know complicate the diagnosis of strangles in horses, Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) and Streptococcus dysgalactiae subsp. equisimilis (S. equisimilis). A total of 18 clinical isolates of S. equi plus the Pinnacle IN vaccine isolate, two isolates of S. zooepidemicus and four isolates of S. equisimilis were obtained via culture and used in the development of the multiplex diagnostic tool. Two multiplex tests were trialed; a conventional multiplex PCR and a real-time multiplex PCR. Both the conventional and real-time multiplex PCR’s were able to distinguish between the streptococci and accurately identified all isolates. However, further testing on 26 field specimens revealed that the real-time multiplex PCR had lower specificity, sensitivity and diagnostic accuracy as compared to the conventional multiplex PCR. This was theorised to be the result of the PEG/KOH solution used in the DNA extraction, possibly interfering with the intercalating dye in the real-time reaction. Based on these preliminary results, the conventional multiplex PCR diagnostic test developed here is recommended for further trials to determine its robustness. The 19 S. equi isolates obtained, including the vaccine, were further subjected to epidemiological studies. These included sequencing of the variable N-terminal region of the antiphagocytic M-protein SeM to determine SeM allele subtypes and a Sau-PCR amplification method, which previously has not been trialled on S. equi isolates. Sau-PCR involves digestion of genomic DNA and subsequent amplification. Two novel strains of S. equi were found within NZ based on the variable region of the seM gene, SeM alleles 99 and 100. SeM allele 100 had a higher pervalance over allele 99 as it was isolated in 6 out of 9 outbreaks and was found to occur on both the North and South Islands of New Zealand. SeM allele 99 was only found to occur on the North Island. Further to this study, the Pinnacle IN vaccine strain, SeM 2 was isolated from lymph node abcesses of two horses. It was unclear as to whether this ‘vaccine breakdown’ was just a severe adverse reaction to the vaccine or if the vaccine reverted to a more virulent type. The Sau-PCR was able to differentiate between the field isolates of S. equi and the vaccine strain but was unable to further differentiate between the field isolates and was therefore determined not as valuable for S. equi epidemiological studies

PPNID : a reference database and molecular identification pipeline for plant-parasitic nematodes

Motivation: The phylum Nematoda comprises the most cosmopolitan and abundant metazoans on Earth and plant-parasitic nematodes represent one of the most significant nematode groups, causing severe losses in agriculture. Practically, the demands for accurate nematode identification are high for ecological, agricultural, taxonomic and phylogenetic researches. Despite their importance, the morphological diagnosis is often a difficult task due to phenotypic plasticity and the absence of clear diagnostic characters while molecular identification is very difficult due to the problematic database and complex genetic background. Results: The present study attempts to make up for currently available databases by creating a manually-curated database including all up-to-date authentic barcoding sequences. To facilitate the laborious process associated with the interpretation and identification of a given query sequence, we developed an automatic software pipeline for rapid species identification. The incorporated alignment function facilitates the examination of mutation distribution and therefore also reveals nucleotide autapomorphies, which are important in species delimitation. The implementation of genetic distance, plot and maximum likelihood phylogeny analysis provides more powerful optimality criteria than similarity searching and facilitates species delimitation using evolutionary or phylogeny species concepts. The pipeline streamlines several functions to facilitate more precise data analyses, and the subsequent interpretation is easy and straightforward

Morphological re-description and molecular identification of Tabanidae (Diptera) in East Africa

Biting flies of the family Tabanidae are important vectors of human and animal diseases across continents. However, records of Africa tabanids are fragmentary and mostly cursory. To improve identification, documentation and description of Tabanidae in East Africa, a baseline survey for the identification and description of Tabanidae in three eastern African countries was conducted. Tabanids from various locations in Uganda (Wakiso District), Tanzania (Tarangire National Park) and Kenya (Shimba Hills National Reserve, Muhaka, Nguruman) were collected. In Uganda, octenol baited F-traps were used to target tabanids, while NG2G traps baited with cow urine and acetone were employed in Kenya and Tanzania. The tabanids were identified using morphological and molecular methods. Morphologically, five genera (Ancala, Tabanus, Atylotus, Chrysops and Haematopota) and fourteen species of the Tabanidae were identified. Among the 14 species identified, six belonged to the genus Tabanus of which two (T. donaldsoni and T. guineensis) had not been described before in East Africa. The greatest diversity of tabanid species were collected from the Shimba Hills National Reserve, while collections from Uganda (around the shores of Lake Victoria) had the fewest number of species. However, the Ancala genus was found in Uganda, but not in Kenya or Tanzania. Maximum likelihood phylogenies of mitochondrial cytochrome c oxidase 1 (COI) genes sequenced in this study show definite concordance with morphological species identifications, except for Atylotus. This survey will be critical to building a complete checklist of Tabanidae prevalent in the region, expanding knowledge of these important vectors of human and animal diseases

Molecular Identification of Eimeria Species in Broiler Chickens in Trinidad, West Indies

Coccidiosis is an intestinal disease of chickens of major economic importance to broiler industries worldwide. Species of coccidia found in chickens include Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox, and Eimeria tenella. In recent years, polymerase chain reaction (PCR) has been developed to provide accurate and rapid identification of the seven known Eimeria species of chickens. The aim of this study was to use species-specific real-time PCR (qPCR) to identify which of the seven Eimeria species are present in Trinidad poultry. Seventeen pooled fecal samples were collected from 6 broiler farms (2–5 pens per farm) across Trinidad. Feces were also collected from birds showing clinical signs of coccidiosis in two live bird markets (pluck shops). qPCR revealed the presence of five species of Eimeria (E. acervulina, E. maxima, E. mitis, E. necatrix, and E. tenella), but not E. brunetti or E. praecox. Mixed infections were detected on all broiler farms, and DNA of two highly pathogenic Eimeria species (E. tenella and E. necatrix) was detected in feces taken from clinically sick birds sampled from the two pluck shops

Molecular Identification of Avian Influenza a Virus in House Flies (Musca Domestica Linnaeus) Collected From Different Poultry Farms in Indonesia

To date, the outbreaks of avian influenza viruses (AIV) in Indonesia are still highly lethal to poultry. The AIV in Indonesia has also infected to humans and make the AIV situation is more serious than in other Asian countries. It is believed that the most likely source of AIV H5N1 infection is the chicken. Since the fly is a wellknown cosmopolitan pest of the poultry farm and home as well as a valuable alternative vehicle of diseasecausing microorganisms, the possibility of the presence of AIV H5N1 in the house flies (Musca domestica L) was investigated with the overall aim of determining the possible mechanical and/or biological vector of avian influenza virus. The RT-PCR was performed on abdominal parts of the house flies. The present of influenza A H5N1 was confirmed by RT-PCR with primers specific for NP, H5 and N1 genes. In the present study, all flies molecularly analyzed have gene expression of avian influenza virus of subtype H5N1. The RT-PCR detects AIV H5N1 isolates of the houseflies samples of the different geographic origin (Sidrap, South Sulawesi, Blitar and Malang, East Java and Karanganyar, Central Java). All those provinces had the different history of the AIV H5N1 outbreaks. On the other hand, results of the RT-PCR assay on the flies collected from Tuban, East Java in the area where never had any AIV outbreaks considered to be negative AIV H5N1 gene expression. Our findings suggest that, in Indonesia, houseflies may be important in the transmission of AIV H5N1. Further studies, however, still need to be done to decide whether houseflies is a mechanical and/or biological vector of avian influenza virus H5N1

Molecular and enological characterization of autochthonous <i>Saccharomyces cerevisiae</i> strains isolated from grape-musts and wines Cannonau

Fermentation by autochthonous yeasts may produce wines with enological properties that are unique of a specific area or variety of grape must. In order to identify yeast starter strains for the production of the Sardinian wine Cannonau DOC, 66 Saccharomyces cerevisiae strains, isolated from musts and wines Cannonau of six vitivinicole areas in Sardinia, were subjected to enological characterization and molecular identification. The RFLP-PCR fingerprinting of the ITS region of rRNA (ITS1-5,8S- ITS2) as well as ethanol, foam, and H2S production were analysed

Molecular identification of different trypanosome species and subspecies in tsetse flies of northern Nigeria

Background: Animal African Trypanosomiasis (AAT) is caused by several species of trypanosomes including Trypanosoma congolense, T. vivax, T. godfreyi, T. simiae and T. brucei. Two of the subspecies of T. brucei also cause Human African Trypanosomiasis. Although some of them can be mechanically transmitted by biting flies; these trypanosomes are all transmitted by tsetse flies which are the cyclical vectors of Trypanosoma congolense, T. godfreyi, T. simiae and T. brucei. We present here the first report assessing the prevalence of trypanosomes in tsetse flies in Nigeria using molecular tools. Methods: 488 tsetse flies of three species, Glossina palpalis palpalis, G. tachinoides and G. morsitans submorsitans were collected from Wuya, Niger State and Yankari National Park, Bauchi State in 2012. Trypanosomes were detected and identified using an ITS1 PCR assay on DNA purified from the ‘head plus proboscis’ (H + P) and abdomen (ABD) parts of each fly. Results: T. vivax and T. congolense Savannah were the major parasites detected. Trypanosomes prevalence was 7.1 % in G. p. palpalis, 11.9 % in G. tachinoides and 13.5 % in G. m. submorsitans. Prevalences of T. congolense Savannah ranged from 2.5 to 6.7 % and of T. vivax were approximately 4.5 %. Trypanosoma congolense Forest, T. godfreyi and T. simiae were also detected in the site of Yankari. The main biological and ecological determinants of trypanosome prevalence were the fly sex, with more trypanosomes found in females than males, and the site, with T. congolense subspp. being more abundant in Yankari than in Wuya. As expected, the trypanosome species diversity was higher in Yankari National Park than in the more agricultural site of Wuya where vertebrate host species diversity is lower. Conclusions: Our results show that T. congolense Savannah and T. vivax are the main species of parasite potentially causing AAT in the two study sites and that Yankari National Park is a potential reservoir of trypanosomes both in terms of parasite abundance and species diversity