scholarly journals DETECTION AND IDENTIFICATION OF YELLOW MOSAIC STUNT DISEASE ON Petunia sp. USING NESTED PCR METHOD

2021 ◽  
Vol 21 (1) ◽  
pp. 56-62
Author(s):  
Suryani Titi Astuti ◽  
Sri Sulandari ◽  
Sedyo Hartono ◽  
Susamto Somowiyarjo
Keyword(s):  
Electron Microscope ◽  
Nested Pcr ◽  
Molecular Detection ◽  
Control Strategies ◽  
Mechanical Transmission ◽  
Chenopodium Amaranticolor ◽  
Virus Particles ◽  
Central Java ◽  
Detection And Identification ◽  
Pcr Method

Yellow mosaic stunt disease was found at some nurseries of Petunia in Sleman, Yogyakarta, also in Muntilan and Magelang Central Java. The disease was very important due to its ability reducing the quality and quantity of Petunia seedlings. The causal agent of the disease may be carried over to imported seeds and necessary to identify as a basic information for developing control strategies. This research was done by mechanical transmission on indicator plants. The observation of the causal agents was conducted using electron microscope with quick dipping method and the molecular detection was done using nested PCR with TobRT up1-TobRT do2 as the external primers and TobN up3-TobN do4 as the internal primers. Mechanical inoculation showed chlorosis symptoms that developed into local spot on Chenopodium amaranticolor as well as mosaic and vein banding on Nicotiana benthamiana. The observation using electron microscope showed rod-shaped virus particles sized approximately 300 nm and by PCR method produced around 568 bp and 400 bp DNA band. Based on the sequence analysis, the disease was caused by Rehmania mosaic virus. This type of Tobamovirus has 96% similarity with ReMV-Japan. ReMV, a plant pathogen which was a member of Tobamovirus that has never been reported in Indonesia. This research was the first report of ReMV in Indonesia infecting Petunia as ornamental plant.

scholarly journals DETECTION AND IDENTIFICATION OF YELLOW MOSAIC STUNT DISEASE ON Petunia sp. USING NESTED PCR METHOD

2021 ◽  
Vol 21 (1) ◽  
pp. 56-62
Author(s):  
Suryani Titi Astuti ◽  
Sri Sulandari ◽  
Sedyo Hartono ◽  
Susamto Somowiyarjo
Keyword(s):  
Electron Microscope ◽  
Nested Pcr ◽  
Molecular Detection ◽  
Ornamental Plant ◽  
Mechanical Transmission ◽  
Chenopodium Amaranticolor ◽  
Virus Particles ◽  
Central Java ◽  
Detection And Identification ◽  
Pcr Method

Detection and identification of yellow mosaic stunt disease on Petunia sp. using nested PCR method. Yellow mosaic stuntdisease was found at some nurseries of Petunia in Sleman, Yogyakarta, also in Muntilan and Magelang Central Java. Thedisease was very important due to its ability reducing the quality and quantity of Petunia seedlings. The causal agent of thedisease may be carried over to imported seeds and necessary to identify as a basic information for developing controlstrategies. This research was done by mechanical transmission on indicator plants. The observation of the causal agents wasconducted using electron microscope with quick dipping method and the molecular detection was done using nested PCRwith TobRT up1-TobRT do2 as the external primers and TobN up3-TobN do4 as the internal primers. Mechanical inoculationshowed chlorosis symptoms that developed into local spot on Chenopodium amaranticolor as well as mosaic and veinbanding on Nicotiana benthamiana. The observation using electron microscope showed rod-shaped virus particles sizedapproximately 300 nm and by PCR method produced around 568 bp and 400 bp DNA band. Based on the sequence analysis,the disease was caused by Rehmania mosaic virus. This type of Tobamovirus has 96% similarity with ReMV-Japan. ReMV, aplant pathogen which was a member of Tobamovirus that has never been reported in Indonesia. This research was the firstreport of ReMV in Indonesia infecting Petunia as ornamental plant.

scholarly journals Detection and Identification of Banana-associated Phytoplasma Using Nested-PCR Method

2019 ◽  
Vol 23 (1) ◽  
pp. 148
Author(s):  
Saurma Mona Astrid Sibarani ◽  
Tri Joko ◽  
Siti Subandiyah
Keyword(s):  
16S Rrna ◽  
Nested Pcr ◽  
Plant Diseases ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Aster Yellows ◽  
Detection And Identification ◽  
Pcr Method ◽  
The 16S Rrna Gene

Phytoplasma is known to be associated with plant diseases in about 300 plant species from various families. Information on the presence of phytoplasma in bananas as one of the pathogens that can cause disease in bananas in Indonesia has never been reported. This research was conducted with the aim to detect the presence of banana phytoplasma by the nested-PCR method and to identify phytoplasma obtained based on the sequence analysis of the 16S rRNA gene. Standard PCR was carried out using P1/P7 primary pairs, followed by nested-PCR using a pair of R16F2n/R16R2m23SR primers separately that could amplify the target 16S rRNA genes in a row at 1600 bp. BLAST analysis shows that the results of phylogenetic analysis of banana phytoplasmic nucleotide cv. manggala from Tasikmalaya and cv. Raja nangka from Banjar has a genetic relationship that is closer to lethal wilt oil palm Phytoplasma (Candidatus Phytoplasma asteris). This phytoplasma belongs to the 16SrI-B group (aster yellows).

Further Observations on the Virus of Epizootic Diarrhea of Infant Mice. An Electron Microscopic Study

1967 ◽  
Vol 25 ◽  
pp. 110-111
Author(s):  
W. G. Banfield ◽  
G. Kasnic ◽  
J. H. Blackwell
Keyword(s):  
Electron Microscope ◽  
Infected Cell ◽  
Microscopic Study ◽  
Intestinal Epithelium ◽  
Ultrastructural Study ◽  
Osmium Tetroxide ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Virus Particles ◽  
Infected Cells

An ultrastructural study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (EDIM virus) was first performed by Adams and Kraft. We have extended their observations and have found developmental forms of the virus and associated structures not reported by them.Three-day-old NLM strain mice were infected with EDIM virus and killed 48 to 168 hours later. Specimens of bowel were fixed in glutaraldehyde, post fixed in osmium tetroxide and embedded in epon. Sections were stained with uranyl magnesium acetate followed by lead citrate and examined in an updated RCA EMU-3F electron microscope.The cells containing virus particles (infected) are at the tips of the villi and occur throughout the intestine from duodenum through colon. All developmental forms of the virus are present from 48 to 168 hours after infection. Figure 1 is of cells without virus particles and figure 2 is of an infected cell. The nucleus and cytoplasm of the infected cells appear clearer than the cells without virus particles.

Electron Microscope Study of RNA's of Paramyxoviruses

1972 ◽  
Vol 30 ◽  
pp. 196-197
Author(s):  
Ruchama Baum ◽  
J.T. Seto
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Electron Microscope Study ◽  
Sendai Virus ◽  
Sodium Dodecyl ◽  
Rna Molecules ◽  
Virus Particles ◽  
Molecular Weights ◽  
Length Measurements

The ribonucleic acid (RNA) of paramyxoviruses has been characterized by biochemical and physiochemical methods. However, paramyxovirus RNA molecules have not been studied by electron microscopy. The molecular weights of these single-stranded viral RNA molecules are not known as yet. Since electron microscopy has been found to be useful for the characterization of single-stranded RNA, this investigation was initiated to examine the morphology and length measurements of paramyxovirus RNA's.Sendai virus Z strain and Newcastle disease virus (NDV), Milano strain, were used. For these studies it was necessary to develop a method of extracting RNA molecules from purified virus particles. Highly purified Sendai virus was treated with pronase (300 μg/ml) at 37°C for 30 minutes and the RNA extracted by the sodium dodecyl sulfate (SDS)-phenol procedure.

scholarly journals INTRACELLULAR FORMS OF POX VIRUSES AS SHOWN BY THE ELECTRON MICROSCOPE (VACCINIA, ECTROMELIA, MOLLUSCUM CONTAGIOSUM)

1953 ◽  
Vol 98 (2) ◽  
pp. 157-172 ◽  
Author(s):  
William H. Gaylord ◽  
Joseph L. Melnick
Keyword(s):  
Electron Microscope ◽  
Hollow Spheres ◽  
Molluscum Contagiosum ◽  
Dense Granule ◽  
Homogeneous Material ◽  
Dense Matrix ◽  
Thin Sections ◽  
Virus Particles ◽  
Typical Inclusion ◽  
Mature Virus

The intracellular development of three pox viruses has been studied with the electron microscope using thin sections of infected tissue. Cells infected with vaccinia, ectromelia, and molluscum contagiosum viruses all form developmental bodies preliminary to the production of mature virus. Developmental bodies, believed to be virus precursors, are round to oval, slightly larger than mature virus particles, less dense to electrons, and have a more varied morphology. It is suggested as a working hypothesis that the process of maturation of a virus particle takes place as follows. In the earliest form the developmental bodies appear as hollow spheres, imbedded in a very dense cytoplasmic mass constituting an inclusion body, or in a less dense matrix near the nucleus in cells without typical inclusion bodies. The spheres become filled with a homogeneous material of low electron density. A small, dense granule appears in each developmental body and grows in size at the expense of the low density material. Following growth of the granule, particles are found with the dimensions of mature virus and having complex internal structure resembling bars or dumbells. Mature virus is ovoid and very dense to electrons. An "empty" interior may be found within its thick walls.

scholarly journals AN ELECTRON MICROSCOPE STUDY OF THE DEVELOPMENT OF A MOUSE HEPATITIS VIRUS IN TISSUE CULTURE CELLS

1965 ◽  
Vol 24 (1) ◽  
pp. 57-78 ◽  
Author(s):  
J. F. David-Ferreira ◽  
R. A. Manaker
Keyword(s):  
Electron Microscope ◽  
Inclusion Bodies ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Dense Material ◽  
Virus Particles ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Tubular Body ◽  
Number Of Particles

Samples taken at different intervals of time from suspension cultures of the NCTC 1469 line of mouse liver—derived (ML) cells infected with a mouse hepatitis virus have been studied with the electron microscope. The experiments revealed that the viruses are incorporated into the cells by viropexis within 1 hour after being added to the culture. An increasing number of particles are found later inside dense cytoplasmic corpuscles similar to lysosomes. In the cytoplasm of the cells from the samples taken 7 hours after inoculation, two organized structures generally associated and never seen in the controls are observed: one consists of dense material arranged in a reticular disposition (reticular inclusion); the other is formed by small tubules organized in a complex pattern (tubular body). No evidence has been found concerning their origin. Their significance is discussed. With the progression of the infection a system of membrane-bounded tubules and cisternae is differentiated in the cytoplasm of the ML cells. In the lumen of these tubules or cisternae, which are occupied by a dense material, numerous virus particles are observed. The virus particles which originate in association with the limiting membranes of tubules and cisternae are released into their lumen by a "budding" process. The virus particles are 75 mµ in diameter and possess a nucleoid constituted of dense particles or rods limiting an electron transparent core. The virus limiting membrane is sometimes covered by an outer layer of a dense material. In the cells from the samples taken 14 to 20 hours after inoculation, larger zones of the cell cytoplasm are occupied by inclusion bodies formed by channels or cisternae with their lumens containing numerous virus particles. In the samples taken 20 hours or more after the inoculation numerous cells show evident signs of degeneration.

scholarly journals Efficient nested-PCR-based method development for detection and genotype identification of Acanthamoeba from a small volume of aquatic environmental sample

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tsui-Kang Hsu ◽  
Jung-Sheng Chen ◽  
Hsin-Chi Tsai ◽  
Chi-Wei Tao ◽  
Yu-Yin Yang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
In Silico ◽  
Environmental Samples ◽  
Method Development ◽  
Small Volume ◽  
Detection Efficiency ◽  
Genotyping Method ◽  
Pcr Method

AbstractAcanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

scholarly journals Molecular Detection and Quantitation of the Red Tide Dinoflagellate Karenia brevis in the Marine Environment

2003 ◽  
Vol 69 (9) ◽  
pp. 5726-5730 ◽  
Author(s):  
M. Gray ◽  
B. Wawrik ◽  
J. Paul ◽  
E. Casper
Keyword(s):  
Real Time ◽  
Marine Environment ◽  
Reverse Transcription ◽  
Molecular Detection ◽  
Red Tide ◽  
Karenia Brevis ◽  
Specific Method ◽  
Reverse Transcription Pcr ◽  
Red Tide Organism ◽  
Pcr Method

ABSTRACT A real-time reverse transcription-PCR method targeting the rbcL gene was developed for the detection and quantitation of the Florida red tide organism, Karenia brevis. The assay was sensitive to less than 1 cell per reaction, did not detect rbcL from 38 nontarget taxa, and accurately quantitated K. brevis organisms in red tide samples from around Florida. These studies have resulted in a sensitive and specific method for K. brevis detection in the marine environment.

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