Molecular Identification Procedures Research Articles

Identification of Distinct Endosymbionts of Whiteflies-infesting Cassava (Manihot esculenta Crantz), using Diagnostic PCR and Sanger Sequencing

The study was conducted during March–December 2021 in Thiruvananthapuram, Kerala for the identification of distinct endosymbionts of cassava whiteflies (Bemisia tabaci (Gennadius) and Aleurodicus dispersus (Russell). Endosymbiotic bacteria found in whiteflies have been reported to interact and provide specific advantages to the hosts and they can be one of the reasons for whiteflies’ insecticidal resistance. In the present study, endosymbiotic bacteria were isolated from the insects, allowed to grow in media after insecticidal treatments and after observing colony morphology, distinct bacterial colonies were sub-cultured and selected for further molecular identification procedures. PCR analysis was carried out for bacteria using 16S rRNA primer, with an annealing temperature of 49°C and yielded fragments at 1500 bp. Based on the sequencing report the observations were that the culturable endosymbionts Providencia present in B. tabaci is resistant to chlorpyrifos and Paenibacillus alvei present in A. dispersus is resistant to imidacloprid and chlorpyrifos. Clostridium senegalens and Acinetobacter are found in control populations of Bemisia and Aleurodicus respectively.The sequences were submitted to NCBI and the accession numbers obtained were OP303253, OP303256, OP303254 and OP295124. Also, through diagnostic PCR using specific bacterial primers, presence of Portiera aleyrodidarum at fragment length of 1000 bp and Wolbachia at 650 bp in B. tabaci and Rickettsia at fragment length of 800 bp in A. dispersus are confirmed. The study could identify bacteria which are decisive in their presence and activity in whiteflies.

EDNA in a bottleneck: Obstacles to fish metabarcoding studies in megadiverse freshwater systems

AbstractThe current capacity of environmental DNA (eDNA) to provide accurate insights into the biodiversity of megadiverse regions (e.g., the Neotropics) requires further evaluation to ensure its reliability for long‐term monitoring. In this study, we first evaluated the taxonomic resolution capabilities of a short fragment from the 12S rRNA gene widely used in fish eDNA metabarcoding studies, and then compared eDNA metabarcoding data from water samples with traditional sampling using nets. For the taxonomic discriminatory power analysis, we used a specifically curated reference dataset consisting of 373 sequences from 258 neotropical fish species (including 47 newly generated sequences) to perform a genetic distance‐based analysis of the amplicons targeted by the MiFish primer set. We obtained an optimum delimitation threshold value of 0.5% due to lowest cumulative errors. The barcoding gap analysis revealed only a 51.55% success rate in species recovery (133/258), highlighting a poor taxonomic resolution from the targeted amplicon. To evaluate the empirical performance of this amplicon for biomonitoring, we assessed fish biodiversity using eDNA metabarcoding from water samples collected from the Amazon (Adolpho Ducke Forest Reserve and two additional locations outside the Reserve). From a total of 84 identified Molecular Operational Taxonomic Units (MOTUs), only four could be assigned to species level using a fixed threshold. Measures of α‐diversity analyses within the Reserve showed similar patterns in each site between the number of MOTUs (eDNA dataset) and species (netting data) found. However, β‐diversity revealed contrasting patterns between the methods. We therefore suggest that a new approach is needed, underpinned by sound taxonomic knowledge, and a more thorough evaluation of better molecular identification procedures such as multi‐marker metabarcoding approaches and tailor‐made (i.e., order‐specific) taxonomic delimitation thresholds.

Molecular identification of bio-fluids in gas phase using infrared spectroscopy.

Bio-fluids are the source of a large number of metabolites. Identification and quantification of them can be an efficient step for understanding the internal chemistry of the body as well as for developing objective diagnostics of diseases. Several techniques have been developed so far; however, their metabolite identification and/or quantification are not reliable enough for acceptance by clinicians. As another promising step in this direction, we push infrared spectroscopy of bio-fluids in gas phase. Here we discuss features of breath and urine headspace realized with Fourier transform infrared spectroscopy. Molecular identification procedures based on component analysis of gas samples are proposed. In this paper, we show that aggregate data from different bio-fluids in gas phase can strengthen the diagnostics of the body state and disease.

Diversity and Biomineralization Potential of the Epilithic Bacterial Communities Inhabiting the Oldest Public Stone Monument of Cluj-Napoca (Transylvania, Romania).

In this study, we investigated the biomineralization potential and diversity of the epilithic bacterial communities dwelling on the limestone statue of Saint Donatus, the oldest public monument of Cluj-Napoca city (Transylvania region, NW Romania). Their spatial distribution together with phylogenetic and metabolic diversity, as well as their capacity to precipitate calcium carbonate was evaluated by combining molecular and phenotypic fingerprinting methods with X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron-microscopy analyses. The results of real-time quantitative PCR, molecular fingerprinting and community-level physiological profiling showed that diverse and abundant bacterial assemblages that differ in relation to their collection site colonized the statue. The cultivation and molecular identification procedures allowed the characterization of 79 bacterial isolates belonging to Proteobacteria (73.4%), Firmicutes (19%), and Actinobacteria (7.6%). Amongst them, the 22 strains identified as being capable of calcium carbonate precipitation were found to belong mostly to Bacillus and Pseudomonas genera. We found that bacteria acted as nucleation sites, inducing the formation of nanoscale aggregates that were shown to be principally composed of vaterite. Furthermore, we expanded the current knowledge on culturable diversity of carbonatogenic bacteria by providing evidence for biogenic vaterite/calcite formation mediated by: Pseudomonas synxantha, P. graminis, Brevibacterium iodinum, Streptomyces albidoflavus, and Stenotrophomonas chelatiphaga. Overall, this study highlights the need to evaluate the carbonatogenetic potential of all the bacterial communities present on stone artwork prior to designing an efficient conservation treatment based on biomineralization.

Variation in the nrDNA ITS sequences of some powdery mildew species: do routine molecular identification procedures hide valuable information?

During the past years, nrDNA ITS sequences have supported the identification of many powdery mildew fungi because comprehensive analyses showed that differences in these sequences have always correlated with the delimitation of different species and formae speciales of the Erysiphales. Published data, obtained using direct sequencing of the PCR products, suggested that even one to five nucleotide differences in the ITS sequences delimit different, albeit closely related, species, and/or indicate differences in host range patterns. Here we show that such differences in the ITS sequences can be detected even in a single sample of a powdery mildew fungus. We sequenced the ITS region in 17 samples, representing six powdery mildew species, both directly and after cloning the PCR products. Among these, samples of O. longipes exhibited two or three, samples of O. neolycopersici three or four, those of an Oidium sp. from Chelidonium majus up to seven, and a sample of another Oidium sp. from Passiflora caerulea two different ITS types determined after cloning. No ITS nucleotide polymorphisms were found in samples of O. lycopersici and Erysiphe aquilegiae. This suggests that some powdery mildew taxa are more variable at the ITS level than others. Thus, although the ITS sequences determined by direct sequencing represent robust data useful in delimitation and phylogenetic analysis of distinct species of the Erysiphales, these need to be used with precaution, and preferably determined after cloning, especially when dealing with closely related taxa at species and sub-species levels. With this method a hitherto undetected genetic diversity of powdery mildews can be revealed.

Mass microscopy: high-resolution imaging mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a technique that localizes the spatial distribution of molecules and identifies structures by their molecular mass signatures. Recently, the resolution of MALDI-IMS has been up to microscopic level. MALDI-IMS does not need either separation or purification procedures of target molecules and enables us to observe the localization of numerous molecules simultaneously. In particular, MALDI-MS time-of-flight/time-of-flight (TOF/TOF) is one of the instruments widely adopted for IMS, which allows the analysis of numerous biomolecules ranging over wide molecular weights. Even in a single data point, hundreds and thousands of mass peaks can be detected, and this makes the resulting mass spectrum extremely complex. This enormous volume of IMS data has driven the development of statistical approaches, especially multivariate analyses. By employing these approaches, researchers can figure out the important characteristics of their IMS data sets. The establishment of automatic molecular identification procedures involving MS(2) analysis, also known as MS/MS, performed by tandem mass spectrometry to obtain the information about molecular structure and composition, and database search available on the web is an important task for the near future. In this review, we introduce IMS-especially MALDI-IMS-with reference to its applications in biomolecular analyses, the workflow of IMS, the principle of IMS and other related technologies.

Isolation and Antifungal Susceptibility of Exophiala dermatitidis Isolates from Human Stool Samples in Nigeria

We screened a total of 460 stool samples from humans with and without underlying disease for the black yeast and neurotrope, Exophiala dermatitidis using conventional and molecular identification procedures. Sixteen (3.5%) samples were positive. Twelve out of the sixteen positive samples where the fungus was recovered were from individuals who had diarrhoea at the moment of isolation. Two asymptomatic positive cases were within the age bracket of 21-30. Susceptibility testing of the strains using the CLSI M38 protocol suggests that almost all the strains were susceptible to amphotericin B, 5 fluorocytosine, itraconazole, fluconazole and voriconazole. Considering the health risk currently posed by this organism in Asia, especially among healthy younger subjects of the population and the scarcity of data on this fungus in Africa in general and Nigeria in particular, some recommendations were suggested.

Tatumella ptyseos, an Unrevealed Causative Agent of Pink Disease in Pineapple

Pink disease is a major problem in the pineapple canning industry. Affected fruit acquire a brownish pigment after pasteurization and can contaminate non-affected fruit before they are released to the consumer. In the last few years, Pantoea citrea has been described as the causative agent of pink disease. In this study, over 300 bacterial isolates from pineapple plants, growing in Mexican commercial fields, and from soil close to plant roots were recovered. Over 250 isolates showed a very high similarity in their phenotypic and genotypic traits with Tatumella ptyseos, a close relative of Pantoea. These isolates exhibited typical pathogenicity reactions in pineapple juice tests, pineapple slices and fruit. On this basis, molecular identification procedures for the Tatumella isolates associated with pink disease were implemented. In affected fruit populations around 10 6 CFU/g of fresh tissue were recovered. This is first time that T. ptyseos is demonstrated as a causal agent of pink disease.

Ribosomal DNA sequencing for identification of aerobic gram-positive rods in the clinical laboratory (an 18-month evaluation).

We have evaluated over a period of 18 months the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic gram-positive rods in the clinical laboratory. Two collections of strains were studied: (i) 37 clinical strains of gram-positive rods well identified by phenotypic tests, and (ii) 136 clinical isolates difficult to identify by standard microbiological investigations, i.e., identification at the species level was impossible. Results of molecular analyses were compared with those of conventional phenotypic identification procedures. Good overall agreement between phenotypic and molecular identification procedures was found for the collection of 37 clinical strains well identified by conventional means. For the 136 clinical strains which were difficult to identify by standard microbiological investigations, phenotypic characterization identified 71 of 136 (52.2%) isolates at the genus level; 65 of 136 (47.8%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 89 of 136 (65.4%) isolates at the species level, 43 of 136 (31.6%) isolates at the genus level, and 4 of 136 (2.9%) isolates at the family level. We conclude that (i) rDNA sequencing is an effective means for the identification of aerobic gram-positive rods which are difficult to identify by conventional techniques, and (ii) molecular identification procedures are not required for isolates well identified by phenotypic investigations.