Species-specific Identification Research Articles

Overview of Lipid Mass Spectrometry and Lipidomics.

Mass spectrometry has played a critical role in the identification and quantitation of lipids present in biological extracts. Various strategies have emerged in order to carry out lipidomic studies. These include both shotgun approaches as well as those engaging liquid chromatographic separation of lipid species prior to mass spectrometric analysis. Nonetheless challenges remain at every level of the lipidomic experiment, including extraction of lipids, identification of specific species, and quantitation of the vast array of lipids present in the sample extract. New strategies have emerged to address some of these issues; however, precise quantitation remains a significant challenge. The use of the ratio of the abundance of the molecular ion species to that of an internal standard enables quite accurate assessment of fold changes within complex lipid species without the need for exact quantitation. Challenges continue to remain in terms of availability of reference standard material as well as relevant internal standards.

Diversity of Nontuberculous Mycobacteria in Kuwait: Rapid Identification and Differentiation of Mycobacterium Species by Multiplex PCR, INNO-LiPA Mycobacteria v2 Assay and PCR Sequencing of rDNA

Objective: Nontuberculous mycobacteria (NTM) often cause disease that is clinically indistinguishable from tuberculosis. Specific identification is important as treatment varies according to Mycobacterium species causing the infection. This study used multiplex PCR (mPCR) assay for rapid differentiation of mycobacterial growth indicator tube 960 system (MGIT) cultures as Mycobacterium tuberculosis (MTB) or NTM together with INNO LiPA Mycobacteria v2 assay (LiPA) and/or PCR sequencing of rDNA for species-specific identification of selected MTB and all NTM isolates in Kuwait. Materials and Methods: DNA was extracted from MGIT cultures (n = 1,033) grown from 664 pulmonary and 369 extrapulmonary specimens from 1,033 suspected tuberculosis patients. mPCR was performed to differentiate MTB from NTM. LiPA was performed and results were interpreted according to kit instructions. rDNA was amplified and sequenced by using panmycobacterial primers. Results: mPCR identified 979 isolates as MTB, 53 as NTM and 1 isolate as mixed culture. LiPA and/or PCR sequencing confirmed 112 of 979 selected isolates as MTB. Mixed culture contained M. tuberculosis and M. fortuitum. LiPA yielded 12 patterns and identified 10 species/species complexes among 47 NTM, M. kansasii + M. scrofulaceum in one culture and 5 isolates only at genus level. PCR sequencing yielded more specific identification for 22 isolates at the species/subspecies level. Conclusions: mPCR rapidly differentiated MTB from NTM. LiPA identified 44 of 52 NTM isolates at the species/species complex level and 2 mixed cultures. PCR sequencing yielded more specific identification at the species/subspecies level. Rapid differentiation as MTB or NTM by mPCR, followed by species-specific NTM identification by LiPA/PCR sequencing is suitable for the proper management of mycobacterial infections in Kuwait.

Identification of Fusarium cf. Verticillioides as The Causal Agent of Pokka Boheng Disease in Sugarcane in the Department of Antioquia, Colombia

The cultivation of sugarcane represents an important part in the Colombian economy due to the diverse applications in industries like liquor, food, paper and biofuels. Sugarcane worldwide production is affected by the presence of phytopathogenic agents, mainly filamentous fungi such as Physalospora tucumanensis (red rot disease) and Fusarium spp. To date in Colombia, Pokka boheng disease whose causal agent is the fungus Fusarium verticillioides, has not been reported, which is why it is necessary to identify appropriately this microorganism, responsible for losses in productivity and food contamination. In order to isolate and identify the infectious agent from symptomatic tissues, disinfection and culture in liquid and solid culture mediums were performed in malt extract (2%) and yeast extract (0.2%) both liquid and solid, for 7 days. After several replicates in agar plate, a purification was made along with a morphological characterization based on the shape and color of the mycelium, as well as the type of spores generated. Additionally, the genetic material was extracted and gene markers (ITS, Elongation Factor 1-α (EF) and β-Tubulin (Btub)) were amplified by PCR. Then, DNA sequencing was used to obtain the data to make a phylogenetic reconstruction by probabilistic methods (Maximum Likelihood and Bayesian Inference). The isolated strain, named as EA-FP0013 was located in the Fujikuroi complex group, with high probable identity to Fusarium verticillioides. Thus, early and species-specific identification of these fungal isolates by molecular methods may allow the timely diagnosis of emerging pathophysiological diseases of interest in the region, and thus propose the respective control strategies.

Prevalence and molecular characterization of zoonotic helminths in dogs.

A cross sectional study was designed to ascertain the prevalence of zoonotic helminths and protozoan parasites in dogs by employing conventional techniques and molecular characterization using polymerase chain reaction (PCR). Companion animals are reservoir hosts for helminths and protozoa species, and cohabitation with humans play a pivotal role in the transmission of zoonotic parasites to humans. A total of 510 faecal samples were collected from pet dogs belongs to various zones of Chennai city, Tamil Nadu, India and were processed by conventional techniques and molecular techniques. Out of 510 dog faeces, 121 samples were found positive for the parasitic eggs, prevalence rate was found to be 23.72%. Species-wise prevalence of the parasite was tabulated. PCR for the species-specific identification of the parasitic eggs was performed. The results revealed 38 samples were positive with product size of 540bp specific for Ancylostoma caninum, 25 samples were positive which yielded a product size of 380bp which is specific for Toxocara canis. None of the faecal samples tested were positive for Echinococcus granulosus which were positive for the presence of Taenia spp. eggs by microscopy. We recommend prevention and control measures focused on improving regular deworming, enhancing awareness of parasitic zoonotic diseases to minimize the transmission risk of parasitic zoonotic diseases from companion animals to humans. PCR can be widely used for species-specific identification of the zoonotic parasites.

PCR-RFLP assays for species-specific identification of fungi belonging to Scopulariopsis and related genera.

Fungi of the Scopulariopsis genus, commonly found in the environment, are opportunistic pathogens that can cause various types of human infections. So far, no efficient molecular method has been developed for species differentiation among Scopulariopsis and related genera. In order to advance this field, we have evaluated performance of polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays, based on cytochrome c oxidase subunit 1 and β-tubulin genes. The assays resulted in 2-10 restriction patterns, depending on the gene amplified and restriction enzyme applied. Pooled analysis of the patterns allowed to propose an algorithm, that can be successfully used for an accurate species-specific identification of 21 species of the Scopulariopsis-like fungi.

Echinostoma revolutum: Development of a high performance DNA-specific primer to demonstrate the epidemiological situations of their intermediate hosts

Echinostomiasis caused by the Echinostoma group, in particular E. revolutum are a significant problem for both humans and other animals. This group has a large number of morphological similarities that are difficult and time-consuming to identify. The present study aimed to develop high-performance tools for the detection of the prevalence of E. revolutum and to reveal the prevalence of E. revolutum infections in intermediate snail hosts in Lopburi province, Thailand. The snail specimens were collected by stratified sampling method and examined to collect trematodes in the larval stage. The specific primer was manually designed and based on 18 s rDNA and verified the specificity and sensitivity for use as an identification tool to compare with classical method, constructed by epidemic mapping. The overall prevalence value of E. revolutum was found to be 16.26%. Tha Luang district had the highest prevalence (70.14%), followed by Chai Badan, Phatthana Nikhom, Tha Wung, Ban Mi, Khok Samrong, Nong Muang and Sa Bot at 42%, 25.14%, 2.52%, 1.73%, 2%, 1.33% and 0.40%, respectively. With regard to the specific primer, it can amplify both cercarial and metacercarial DNA (90 pg/μl.) and discriminated E. revolutum from its hosts, other trematodes and other echinostome larvae with no cross-reactions. Therefore, the developed specific primer can be used as a species-specific identification tool with a high degree of sensitivity and specificity. Consequently, this data is important for monitoring the outbreak of E. revolutum. It can be applied for initiating surveillance programs of snail-borne diseases in both medical and veterinary studies.

First record of the semi-slug Omalonyx unguis (d’Orbigny, 1837) (Gastropoda, Succineidae) in the Misiones Province, Argentina

Omalonyx unguis (d’Orbigny, 1837) is a semi-slug inhabiting the Paraná river basin. This species belongs to Succineidae, a family comprising a few representatives in South America. In this work, we provide the first record for the species from Misiones Province, Argentina. Previous records available for Omalonyx in Misiones were identified to the genus level. We examined morphological characteristics of the reproductive system and used DNA sequences from cytochrome oxidase subunit I (COI) gene for species-specific identification. These new distributional data contribute to consolidate the knowledge of the molluscan fauna in northeastern Argentina.

Proteomic and molecular fingerprinting for identification and tracking of fish pathogenic Streptococcus

In this study, molecular and proteomic methods were compared with serological ones for species-specific identification and typing of S. parauberis and S. iniae isolated from fish. Slide agglutination, dot-blot, repetitive sequence-based PCR (REP-PCR) DNA fingerprinting and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were performed. A new serotype was defined for S. parauberis isolated from turbot based on proteomic and serological results. Genus and/or species-specific protein biomarkers were found for the Streptococcus strains tested using MALDI-TOF-MS. Protein patterns specific to serotype and subserotype were also observed for S. parauberis strains. Species- and strain-specific DNA profiles were also obtained by REP-PCR. A good correlation was found between serological, proteomic and molecular methods for species differentiation and strain typing. However, the accuracy and discriminatory power of the MALDI-TOF-MS make it the most promising tool for the diagnosis of streptococcosis in fish and serotyping of bacterial fish pathogens belonging to the species S. parauberis, avoiding the use of experimental animals. Also highlights its usefulness for rapid and cost-effective routine application in veterinary laboratories.

Filling the gaps in clinical proteomics: a do-it-yourself guide for the identification of the emerging pathogen Arcobacter by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Arcobacters are considered emerging gastrointestinal pathogens. Rapid, reliable and species-specific identification of these bacteria is important. Biochemical tests commonly yield negative or variable results. Molecular methods prove more reliable but are time consuming and lack specificity. Matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a fast, cheap and robust technique that has revolutionized genus and species identification in clinical microbiology.The performance of an in vitro diagnostic (RUO) spectral database of MALDI-TOF MS for the identification of human clinically relevant Arcobacter isolates was validated and compared to an in house created Reference Spectral database (RS) containing a representative set of deposited Arcobacter strains of zoonotic interest. A challenge panel of clinical, human and veterinary, unique Campylobacteraceae strains was used to test accuracy.Using direct colony transfer, sensitivity with RS was significantly better than with RUO for A. butzleri and A. cryaerophilus identification (100% and 92% versus 74% and 16%). For A. skirrowii, sensitivity remained low (21% versus 0%). Reanalysis using formic acid overlay (on-target extraction) augmented sensitivity for the latter species to 64%. Specificity of RS database remained excellent without any misidentifications of human clinical strains including Campylobacter fetus and C. jejuni/coli.The use of an enriched database for MALDI-TOF MS identification of Arcobacter spp. of human interest produced high-confidence identifications to species level resulting in a significantly improved sensitivity with conservation of excellent specificity. Misidentifications, which can have therapeutic and public health consequences, were not encountered.

PENGUJIAN PENANDA JENIS SPESIFIK PADA JAMUR YANG BERPOTENSI SEBAGAI AGENS PENGENDALI HAYATI PENYAKIT BUSUK AKAR PADA AKASIA

Root rot caused by Ganoderma philippii (Bres. & Henn. ex Sacc.) Bres., is an important disease in Acacia plantation. A strategy to control this disease is currently being developed, particularly on the application of biological control agent (BCA). Species specific primers for rapid identification of potential fungi as BCA were developed. This study aimed to obtain the best DNA condition and the best primers for species specific identification. DNA with 20x dilution is the best condition for amplification of the ITS fragment thus used for rapid species identification. The best primer set to detect Cerrena sp. is CrF1/CrR1, PbF2/PbR2 is the best primer set for Phlebiopsis sp. 1. There was no specific primer suitable to detect Phlebia sp. 1 and Phlebia sp. 2 only but Pl-2F1/Pl-2R4 is the best primer set for Phlebia spp.

Real-time PCR, a great tool for fast identification, sensitive detection and quantification of important plant-parasitic nematodes

Plant-parasitic nematodes can cause significant damage to agricultural crops and forests worldwide, resulting in major economic losses. Some nematode species do not occur in all areas and are regulated as quarantine organisms. To avoid introduction and spread of these organisms, fast, simple and reliable detection and identification methods are needed, that help plant diagnostic services such as reference centres or national plant protection organizations (NPPOs) to rapidly identify suspicious nematodes. Real-time PCR is one of the fastest, most sensitive and reliable methods to fulfil this task. It is a DNA-based method that is easy to learn with the only requirement of having a specific thermocycler (Real-time Platform) and the appropriate chemistry. Real-time PCR provides very sensitive detection and species-specific identification with the potential to quantify target organisms if required. Following DNA extraction, results can be seen in 1–3 h and management decisions applied. Real-time PCR can be used for high-throughput analysis of many samples and in some cases for multiplexing, allowing for identification of more than one species in a single reaction. Over the past 15 years, real-time PCR methods have been developed for the main plant-parasitic nematodes, in particular the regulated species. This paper reviews the achievements in plant nematology diagnostics using real-time PCR as the method of choice for fast and reliable detection, identification and even quantification of plant parasitic nematodes.

Rapid identification of bovine mastitis pathogens by MALDI-TOF Mass Spectrometry

ABSTRACT: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been shown to be an alternative method for identification of bacteria via their protein profile spectra, being able to identify bacteria at the genus, species and even at subspecies level. With the aim of large-scale identification of pathogens causing mastitis by this platform, a total of 305 isolates of bacteria identified from cows with subclinical mastitis were analyzed by conventional microbiological culture (MC) as well as by MALDI-TOF MS coupled with Biotyper data processing. Approximately 89% of the identifications performed by MALDI-TOF MS were consistent with results obtained by MC. From the remaining isolates (11%), 6.3% of isolates were classified as misidentified (discordance for both genus and species level), and 4.7% showed identification agreement at the genus level but not at the species level, being classified as unidentified at species level. The disagreement results were mostly associated with identification of Streptococcus and Enterococcus species probably due to the narrow phenotypic similarity between these two genera. These disagreement results suggest that biochemical assays might be prone to identification errors and, MALDI-TOF MS therefore may be an alternative to overcome incorrect species-specific identification. Standard microbiological methods for bovine mastitis diagnosis are time consuming, laborious and prone to errors for some bacteria genera. In our study, we showed that MALDI-TOF MS coupled with Biotyper may be an alternative method for large-scale identification of bacteria isolated from milk samples compared to classical microbiological routine protocols.

Species-specific detection of Candida tropicalis using evolutionary conserved intein DNA sequences

Development of molecular markers for specific detection of microbial pathogens using real-time polymerase chain reaction (PCR) is an appealing and challenging technique. A real-time PCR is an emerging technology frequently used to detect the aetiologic agents. In recent times, designing species-specific primers for pathogen detection is gaining momentum. The method offers rapid, accurate and cost-effective strategy to identify the target, thus providing sufficient time to instigate appropriate chemotherapy. The study highlights the use of intein DNA sequence as molecular markers for species-specific identification of Candida tropicalis. The study also offers a prototype model for developing multifaceted PCR assays using intein DNA sequences, and provides a developmental starting point for point-of-care testing in near future.

Description and Identification of Four Species of Plant-parasitic Nematodes Associated with Forage Legumes

Four species of plant parasitic nematodes were present in soil samples planted with forage legumes at Alachua County, Florida, USA. The detected species Belonolaimus longicaudatus, Criconemella ornate, Hoplolaimus galeatus, and Paratrichodorus minor were described in the present study. They belong to orders Rhabditida (Belonolaimus longicaudatus, Criconemella ornate, and Hoplolaimus galeatus) and Triplonchida (Paratrichodorus minor) and to taxonomical families Dolichodoridae (Belonolaimus longicaudatus), Hoplolaimidae (Hoplolaimus galeatus) Criconematidae (Criconemella ornate), and Trichodoridae (Paratrichodorus minor). The identification of the present specimens was based on the classical taxonomy, following morphological and morphometrical characters in the species specific identification keys

Identification of Five Phytonematode Species Associated with Forage Legumes

Five species of plant parasitic nematodes were present in soil samples planted with forage legumes at Florida, USA. The found species Meloidogyne arenaria, M. incognita, M. javanica, Pratylenchus brachyurus and Tylenchorhynchus martini were depicted in this study. These species belong to order Rhabditida and to taxonomical families Hoplolaimidae (subfamily Meloidogyninae: Meloidogyne arenaria, M. incognita and M. javanica), Pratylenchidae (Pratylenchus brachyurus) and Dolichodoridae (Tylenchorhynchus martini). The identification of the processed specimens was based on the classical taxonomy, following perineal pattern characters for species of Meloidogyne as well as morphological and morphometrical characters in the species specific identification keys for Pratylenchus brachyurus and Tylenchorhynchus martin

Genetic heterogeneity among Vibrio alginolyticus strains, and design of a PCR-based identification method using gyrB gene sequence.

Vibrio alginolyticus, a pathogen among humans and marine animals, is ubiquitous in marine environments. The aims of this study were to analyze the relationships between genetic diversity and origins, and to develop new primers based on the gyrB sequence to identify V. alginolyticus isolated from various sources. To determine the genetic diversity of this bacterium, an arbitrarily primed polymerase chain reaction (AP-PCR) technique was performed on 36 strains of V. alginolyticus isolated from diarrhea patients and from diseased marine animals and environments in southern Thailand. The results showed distinct DNA fingerprints of all strains, indicating that they are genetically heterogeneous. For species-specific identification of V. alginolyticus, primers targeting the gyrB gene of V. alginolyticus were developed. Thirty reference Vibrio spp., 13 non-Vibrio spp., and 160 strains of V. alginolyticus isolated from various sources in southern Thailand were used to evaluate the specificity of these primers. Our results showed that the gyrB primers could specifically identify V. alginolyticus from all sample types. In addition, the detection limit of the PCR was at least 95 pg of DNA template. Therefore, we concluded that the newly designed gyrB primers are rapid, highly sensitive, and specific to identify V. alginolyticus isolated from various sources.

MALDI-TOF-MS based identification and molecular characterization of food associated methicillin-resistant Staphylococcus aureus

Food-borne methicillin resistant Staphylococcus aureus (MRSA) is involved in two-fold higher mortality rate compared to methicillin susceptible S. aureus (MSSA). Eventhough Mysuru recognized as cleanest city in the world, prevalence of food contamination is not detailed. The aim is to screen food samples from Mysuru area and to characterize MRSA strain, employing MALDI-Biotyper, multiplex PCR to distinguish between MRSA and MSSA by PCR-coupled single strand conformation polymorphism (PCR-SSCP). Of all the food-borne pathogens, S. aureus contamination accounts for 94.37 ± 0.02% (P < 0.01), strains characterized by means of nuc genes, followed by species specific identification by Coa, Eap and SpA genes and multiplex PCR to confirm the presence of three methicillin resistant staphylococcal species simultaneously using nuc and phoP genes. Amplification of mecA gene in 159 isolates confirmed that all strains are methicillin resistant, except UOM160 (MSSA) and multi-drug resistant (MDR) in 159 isolates confirmed by 22 sets of β-lactam antibiotics. MSSA and MDR-MRSA were discriminated by PCR-SSCP using nuc gene for the first time. From the present studies, compared to conventional methods MALDI-Biotyper emerged as an effective, sensitive (>99%), robust (<2 min), and alternative tool for pathogen identification, and we developed a PCR-SSCP technique for rapid detection of MSSA and MRSA strains.

Interlaboratory validation of an improved method for detection of Cyclospora cayetanensis in produce using a real-time PCR assay

A collaborative validation study was performed to evaluate the performance of a new U.S. Food and Drug Administration method developed for detection of the protozoan parasite, Cyclospora cayetanensis, on cilantro and raspberries. The method includes a sample preparation step in which oocysts are recovered from produce using an enhanced produce washing solution containing 0.1% Alconox and a commercially available method to disrupt the C. cayetanensis oocysts and extract DNA. A real-time PCR assay targeting the C. cayetanensis 18S rDNA gene with an internal amplification control to monitor PCR inhibition provides species-specific identification. Five laboratories blindly analyzed a total of 319 samples consisting of 25 g of cilantro or 50 g of raspberries which were either uninoculated or artificially contaminated with C. cayetanensis oocysts. Detection rates for cilantro inoculated with 200, 10, and 5 oocysts, were 100%, 80%, and 31%, respectively. For raspberries, the detection rates for samples inoculated with 200, 10, and 5 oocysts were 100%, 90% and 50%, respectively. All uninoculated samples, DNA blank extracts, and no-template PCR controls were negative. Reproducibility between laboratories and analysts was high and the method was shown to be an effective analytical tool for detection of C. cayetanensis in produce.

Standardization of a Two-step Real-time Polymerase Chain Reaction Based Method for Species-specific Detection of Medically Important Aspergillus Species

Purpose: Standardization of Aspergillus polymerase chain reaction (PCR) poses two technical challenges (a) standardization of DNA extraction, (b) optimization of PCR against various medically important Aspergillus species. Many cases of aspergillosis go undiagnosed because of relative insensitivity of conventional diagnostic methods such as microscopy, culture or antigen detection. The present study is an attempt to standardize real-time PCR assay for rapid sensitive and specific detection of Aspergillus DNA in EDTA whole blood. Materials and Methods: Three nucleic acid extraction protocols were compared and a two-step real-time PCR assay was developed and validated following the recommendations of the European Aspergillus PCR Initiative in our setup. In the first PCR step (pan-Aspergillus PCR), the target was 28S rDNA gene, whereas in the second step, species specific PCR the targets were beta-tubulin (for Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus), gene and calmodulin gene (for Aspergillus niger). Results: Species specific identification of four medically important Aspergillus species, namely, A. fumigatus, A. flavus, A. niger and A. terreus were achieved by this PCR. Specificity of the PCR was tested against 34 different DNA source including bacteria, virus, yeast, other Aspergillus sp., other fungal species and for human DNA and had no false-positive reactions. The analytical sensitivity of the PCR was found to be 102 CFU/ml. Conclusion: The present protocol of two-step real-time PCR assays for genus- and species-specific identification for commonly isolated species in whole blood for diagnosis of invasive Aspergillus infections offers a rapid, sensitive and specific assay option and requires clinical validation at multiple centers.

Nematode Species Identification-Current Status, Challenges and Future Perspectives for Cyathostomins.

Human and animal health is globally affected by a variety of parasitic helminths. The impact of co-infections and development of anthelmintic resistance requires improved diagnostic tools, especially for parasitic nematodes e.g., to identify resistant species or attribute pathological effects to individual species or particular species combinations. In horses, co-infection with cyathostomins is rather a rule than an exception with typically 5 to 15 species (out of more than 40 described) per individual host. In cyathostomins, reliable morphological species differentiation is currently limited to adults and requires highly specialized expertize while precise morphological identification of eggs and early stage larvae is impossible. The situation is further complicated by a questionable validity of some cyathostomins while others might actually represent cryptic species complexes. Several molecular methods using different target sequences were established to overcome these limitations. For adult worms, PCR followed by sequencing of mitochondrial genes or external or internal ribosomal RNA spacers is suitable to genetically confirm morphological identifications. The most commonly used method to differentiate eggs or larvae is the reverse-line-blot hybridization assay. However, both methods suffer from the fact that target sequences are not available for many species or even that GenBank® entries are unreliable regarding the cyathostomin species. Recent advances in proteomic tools for identification of metazoans including insects and nematodes of the genus Trichinella will be evaluated for suitability to diagnose cyathostomins. Future research should focus on the comparative analysis of morphological, molecular and proteomic data from the same cyathostomin specimen to optimize tools for species-specific identification.