Development of Molecular Identification Method for Genus Alexandrium (Dinophyceae) Using Whole-Cell FISH

Estimation of postmortem interval (PMI) is paramount in modern forensic investigation. After the disappearance of the early postmortem phenomena conventionally used to estimate PMI, entomologic evidence provides important indicators for PMI estimation. The age of the oldest fly larvae or pupae can be estimated to pinpoint the time of oviposition, which is considered the minimum PMI (PMImin). The development rate of insects is usually temperature dependent and species specific. Therefore, species identification is mandatory for PMImin estimation using entomological evidence. The classical morphological identification method cannot be applied when specimens are damaged or have not yet matured. To overcome this limitation, some investigators employ molecular identification using mitochondrial cytochrome c oxidase subunit I (COI) nucleotide sequences. The molecular identification method commonly uses Sanger’s nucleotide sequencing and molecular phylogeny, which are complex and time consuming and constitute another obstacle for forensic investigators. In this study, instead of using conventional Sanger’s nucleotide sequencing, single-nucleotide polymorphisms (SNPs) in the COI gene region, which are unique between fly species, were selected and targeted for single-base extension (SBE) technology. These SNPs were genotyped using a SNaPshot® kit. Eleven Calliphoridae and seven Sarcophagidae species were covered. To validate this genotyping, fly DNA samples (103 adults, 84 larvae, and 4 pupae) previously confirmed by DNA barcoding were used. This method worked quickly with minimal DNA, providing a potential alternative to conventional DNA barcoding. Consisting of only a few simple electropherogram peaks, the results were more straightforward compared with those of the conventional DNA barcoding produced by Sanger’s nucleotide sequencing.

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Molecular Identification of the Toxic Alexandrium tamiyavanichii (Dinophyceae) by the Whole-cell FISH Method

Fisheries and aquatic sciences ◽

10.5657/fas.2004.7.4.175 ◽

2004 ◽

Vol 7 (4) ◽

pp. 175-183 ◽

Cited By ~ 1

Author(s):

Choong-Jae Kim ◽

Sada-Akfi Yoshimatsu ◽

Yoshihiko Sako ◽

Chang-Hoon Kim

Keyword(s):

Molecular Identification ◽

Whole Cell ◽

Fish Method

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Molecular Identification of Lipase LipA from Pseudomonas protegens Pf-5 and Characterization of Two Whole-Cell Biocatalysts Pf-5 and Top10lipA

Journal of Microbiology and Biotechnology ◽

10.4014/jmb.1312.12005 ◽

2014 ◽

Vol 24 (5) ◽

pp. 619-628 ◽

Cited By ~ 12

Author(s):

Daiming Zha ◽

Li Xu ◽

Houjin Zhang ◽

Yunjun Yan

Keyword(s):

Molecular Identification ◽

Whole Cell

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Whole-cell spectroscopy is a convenient tool to assist molecular identification of cultivatable marine bacteria and to investigate their adaptive metabolism

Talanta ◽

10.1016/j.talanta.2009.10.020 ◽

2010 ◽

Vol 80 (5) ◽

pp. 1758-1770 ◽

Cited By ~ 30

Author(s):

Stéphanie Salaün ◽

Nelly Kervarec ◽

Philippe Potin ◽

Dominique Haras ◽

Martial Piotto ◽

...

Keyword(s):

Molecular Identification ◽

Marine Bacteria ◽

Whole Cell ◽

Convenient Tool

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Validation of an Effective Protocol for Culicoides Latreille (Diptera: Ceratopogonidae) Detection Using eDNA Metabarcoding

Insects ◽

10.3390/insects12050401 ◽

2021 ◽

Vol 12 (5) ◽

pp. 401

Author(s):

Yoamel Milián-García ◽

Lauren A. A. Janke ◽

Robert G. Young ◽

Aruna Ambagala ◽

Robert H. Hanner

Keyword(s):

Molecular Identification ◽

Molecular Detection ◽

Morphological Characteristics ◽

Species Level ◽

Morphological Identification ◽

Culicoides Species ◽

Nacl Solution ◽

Identification Method

eDNA metabarcoding is an effective molecular-based identification method for the biosurveillance of flighted insects. An eDNA surveillance approach maintains specimens for secondary morphological identification useful for regulatory applications. This study identified Culicoides species using eDNA metabarcoding and compared these results to morphological identifications of trapped specimens. Insects were collected using ultraviolet (UV) lighted fan traps containing a saturated salt (NaCl) solution from two locations in Guelph, Ontario, Canada. There were forty-two Culicoides specimens collected in total. Molecular identification detected four species, C. biguttatus, C. stellifer, C. obsoletus, and C. mulrennani. Using morphological identification, two out of these four taxonomic ranks were confirmed at the species level (C. biguttatus and C. stellifer) and one was confirmed at the subgenus level (Avaritia [C. obsoletus]). No molecular detection of Culicoides species occurred in traps with an abundance of less than three individuals per taxon. The inconsistency in identifying Culicoides specimens to the species level punctuates the need for curated DNA reference libraries for Culicoides. In conclusion, the saturated salt (NaCl) solution preserved the Culicoides’ morphological characteristics and the eDNA.

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Sponge-Cell Culture? A Molecular Identification Method for Sponge Cells

Marine Biotechnology ◽

10.1007/s10126-002-0090-1 ◽

2003 ◽

Vol 5 (5) ◽

pp. 443-449 ◽

Cited By ~ 13

Author(s):

Detmer Sipkema ◽

Hans G.H.J. Heilig ◽

Antoon D.L. Akkermans ◽

Ronald Osinga ◽

Johannes Onji ◽

...

Keyword(s):

Cell Culture ◽

Molecular Identification ◽

Sponge Cell ◽

Identification Method

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Electron microscopy in diagnostic virology

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100110702 ◽

1984 ◽

Vol 42 ◽

pp. 70-73

Author(s):

S. E. Miller

Keyword(s):

Electron Microscopy ◽

Tissue Culture ◽

Molecular Identification ◽

Point Of View ◽

Negative Stain ◽

Viral Diagnosis ◽

Electron Microscopist ◽

Diagnostic Virology

The techniques for detecting viruses are many and varied including FAT, ELISA, SPIRA, RPHA, SRH, TIA, ID, IEOP, GC (1); CF, CIE (2); Tzanck (3); EM, IEM (4); and molecular identification (5). This paper will deal with viral diagnosis by electron microscopy and will be organized from the point of view of the electron microscopist who is asked to look for an unknown agent--a consideration of the specimen and possible agents rather than from a virologist's view of comparing all the different viruses. The first step is to ascertain the specimen source and select the method of preparation, e. g. negative stain or embedment, and whether the sample should be precleared by centrifugation, concentrated, or inoculated into tissue culture. Also, knowing the type of specimen and patient symptoms will lend suggestions of possible agents and eliminate some viruses, e. g. Rotavirus will not be seen in brain, nor Rabies in stool, but preconceived notions should not prejudice the observer into missing an unlikely pathogen.

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HVEM Analysis of Microfilament Patterns in The Margins of Tissue Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600003512 ◽

1980 ◽

Vol 38 ◽

pp. 808-811

Author(s):

Carol Allen

Keyword(s):

Cell Movement ◽

Cell Spreading ◽

Living Cells ◽

Tissue Cell ◽

Large Sample Size ◽

Cell Periphery ◽

Whole Cell ◽

Critical Point Drying ◽

Lamellar Region ◽

Tissue Cells

When provided with a suitable solid substrate, tissue cells undergo a rapid conversion from the spherical form expressed in suspension culture to a characteristic flattened morphology. As a result of this conversion, called cell spreading, the cell nucleus and organelles come to occupy a central region of “deep cytoplasm” which slopes steeply into a peripheral “lamellar” region less than 1 pm thick at its outer edge and generally free of cell organelles. Cell spreading is accomplished by a continuous outward repositioning of the lamellar margins. Cell translocation on the substrate results when the activity of the lamellae on one side of the cell become dominant. When this occurs, the cell is “polarized” and moves in the direction of the “leading lamellae”. Careful analysis of tissue cell locomotion by time-lapse microphotography (1) has shown that the deformational movements of the leading lamellae occur in a repeating cycle of advance and retreat in the direction of cell movement and that the rate of such deformations are positively correlated with the speed of cell movement. In the present study, the physical basis for these movements of the cell margin has been examined by comparative light microscopy of living cells with whole-mount electron microscopy of fixed cells. Ultrastructural observations were made on tissue cells grown on Formvar-coated grids, fixed with glutaraldehyde, further processed by critical-point drying, and then photographed in the High Voltage Electron Microscope. This processing and imaging system maintains the 3-dimensional organization of the whole cell, the relationship of the cell to the substrate, and affords a large sample size which facilitates quantitative analysis. Comparative analysis of film records of living cells with the whole-cell micrographs revealed that specific patterns of microfilament organization consistently accompany recognizable stages of lamellar formation and movement. The margins of spreading cells and the leading lamellae of locomoting cells showed a similar pattern of MF repositionings (Figs. 1-4). These results will be discussed in terms of a working model for the mechanics of lamellar motility which includes the following major features: (a) lamellar protrusion results when an intracellular force is exerted at a locally weak area of the cell periphery; (b) the association of cortical MFs with one another determines the local resistance to this force; (c) where MF-to-MF association is weak, the cell periphery expands and some cortical MFs are dragged passively forward; (d) contact of the expanded area with the substrate then triggers the lateral association and reorientation of these cortical MFs into MF bundles parallel to the direction of the expansion; and (e) an active interaction between these MF bundles associated with the cortex of the expanded lamellae and the cortical MFs which remained in the sub-lamellar region then pulls the latter MFs forward toward the expanded area. Thus, the advance of the cell periphery on the substrate occurs in two stages: a passive phase in which some cortical MFs are dragged outward by the force acting to expand the cell periphery, and an active phase in which additional cortical MFs are pulled forward by interaction with the first set. Subsequent interactions between peripheral microfilament bundles and filaments in the deeper cytoplasm could then transmit the advance gained by lamellar expansion to the bulk of the cytoplasm.

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