nucleotide sequence analysis
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Author(s):  
Neval Berrin Arserim ◽  
Metin Gürçay ◽  
Ahmed Sait ◽  
Mustafa Türkdoğan

Background: In this study, partial nucleotide sequence analysis of the G gene was performed for the molecular characterization of the virus that caused the bovine ephemeral fever virus (BEFV) epidemic in Turkey in 2020. Phylogenetic analysis of these nucleotide sequences was performed with the virus nucleotide sequences of the epidemics seen in 2008 and 2012. These sequences were announced in GenBank. Phylogenetic analysis of these nucleotide sequences was performed with the virus nucleotide sequences of the epidemics seen in 2008 and 2012. Methods: The study was conducted in dairy cattle holdings located in Diyarbakır Sur, Çınar and Dicle regions in South-eastern Turkey in August-November 2020. The number of animals in the holdings consisted of 750 (n=750), 150 (n=150) and 200 (n=200) cattle, respectively. Result: Severe respiratory symptoms and high mortality in the affected animals were notable symptoms. As a result of the phylogenetic analysis, it was determined that the virus that caused the epidemic in Turkey in 2020 was formed by a new variant in the Turkey-2 group, which was similar to the Indian isolates, unlike the Turkey-1 group, which was close to the Middle East variants in 2008 and 2012 isolates.


2021 ◽  
Author(s):  
Go-Eun Shin ◽  
Ji-Young Park ◽  
Kyoung-Ki Lee ◽  
Mi-Kyeong Ko ◽  
Bok-Kyung Ku ◽  
...  

Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses in the global swine industry. Frequent genetic variations in this virus cause difficulties in controlling and accurately diagnosing PRRSV. Methods In this study, we investigated the genetic characteristics of PRRSV-1 and PRRSV-2 circulating in Korea from January 2018 to September 2021 and evaluated three one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Results A total of 129 lung samples were collected, consisting of 47 samples for PRRSV-1, 62 samples for PRRSV-2, and 20 PRRSV-negative samples. Nucleotide sequence analysis of open reading frames (ORFs) 5, ORF6, and ORF7 genes from PRRSV samples showed that PRRSV-1 belonged to subgroup A (43/47, 91.49%) and subgroup C (4/47, 8.51%), whereas PRRSV-2 was classified as lineage 1 (25/62, 40.32%), Korean lineage (Kor) C (13/62, 20.97%), Kor B (10/62, 16.13%), lineage 5 (9/62, 14.52%), and Kor A (5/62, 8.06%). Amino acid sequence analysis showed that the neutralizing epitope and T cell epitope of PRRSV-1, and the decoy epitope region and hypervariable regions of PRRSV-2 had evolved under positive selection pressure. In particular, the key amino acid substitutions were found at positions 102 and 104 of glycoprotein 5 (GP5) in some PRRSV-2, and at positions 10 and 70 of membrane protein (M) in most PRRSV-2. In addition, one-step real-time RT-PCR assays, comprising two commercial tests and one test recommended by the World Organization for Animal Health (OIE), were evaluated. Conclusion The results revealed that two of the real-time RT-PCR assays had high sensitivities and specificities, whereas the real-time RT-PCR assay of the OIE had low sensitivity due to mismatches between nucleotides of Korean PRRSVs and forward primers. In this study, we genetically characterized recent PRRSV occurrences and evaluated three one-step real-time RT-PCR assays used in Korea.


2021 ◽  
Vol 26 (2(49)) ◽  
pp. 73-85
Author(s):  
Yu. A. Popovych ◽  
O. M. Blagodarova ◽  
S. V. Chebotar

Introduction. Gliadins are monomeric and highly polymorphic storage proteins of wheat endosperm, which together with glutenins form a gluten complex that determines the breadmaking properties of wheat. Allelic variants of gliadins are an important feature in the selection of material for breeding, but their determination by electrophoresis in acid PAGE is quite difficult. Aim. The aim of this study was to investigate the polymorphism of the Taglgap microsatellite locus and to analyze its correspondence to the polymorphism of allelic variants of gliadins that have been revealed by acid PAGE electrophoresis. Methods. 140 cultivars and lines of bread wheat of Ukrainian and foreign selection were analyzed. Electrophoresis of storage proteins was performed in an acid PAGE according to the method of F. O. Poperellia (1989), allelic variants were designated according to the international nomenclature (Metakovsky et al., 2018). DNA was isolated by CTAB method and PCR was performed with primers to the Taglgap microsatellite (Devos et al., 1995). PCR products were fractionated in 7% PAGE and stained with silver staining method. Nucleotide sequences were searched by BLAST and aligned by MAFT methods. The main results. 19 allelic variants of gliadins and 11 alleles of the Taglgap locus were identified. In the collection of Ukrainian varieties there were Gli-B1b, Gli-B1c, Gli-B1d, Gli-B1e, Gli-B1f, Gli-B1g, Gli-B1h, Gli-B1l and Gli-B1o allelic variants and alleles of Taglgap 216 bp, 237 bp, 246 bp, 248 bp, 252 bp, 267 bp, 270 bp and null. In the foreign collection of varieties − Gli-B1a, Gli-B1b, Gli-B1c, Gli-B1d, Gli-B1e, Gli-B1f, Gli-B1g, Gli-B1h, Gli-B1i, Gli-B1j, Gli-B1k, Gli -B1l, Gli-B1m, Gli-B1n, Gli-B1o, Gli-B1p, Gli-B1q, Gli-B1r, Gli-B1s and 213 bp, 216 bp, 237 bp, 246 bp, 248 bp, 250 bp, 252 bp, 270 bp, 285 bp and null. Nucleotide sequence analysis in the NCBI database showed the presence of a number of other alleles of the Taglgap microsatellite not only in bread wheat but also in some species of the Triticum L. and Aegilops L. genus. Conclusions. The detected polymorphism correlates with the polymorphism of allelic variants of gliadins of Gli-B1 locus and makes it possible to identify Gli-B1a, Gli-B1d, Gli-B1h and Gli-B1l allelic variants, and for Ukrainian varieties with high probability also Gli-B1b allelic variant. However, this marker does not allow identifying Gli-B1c, which is important for selection.


Author(s):  
N. G. Ogbuji ◽  
A. E. Ataga ◽  
P. M. Tari-Ukuta ◽  
C. J. Olisedeme

Aims: A study was conducted to identify fungal species isolated from dumpsite soil in University of Port Harcourt using molecular techniques. Methodology: Molecular methods for determining the species of a fungus based on the amplification and sequencing of the internal subscribed spacer (ITS) region of the fungal rRNA operon using molecular markers was applied. Soil sample was collected from a dumpsite in the University of Port Harcourt, Rivers State, Nigeria. Isolation of fungi associated with the dumpsite soil was carried out using spread plate method. Fungal genomic DNA was extracted using Quick-DNA Fungal/Bacterial Miniprep kit. The ITS1-2 gene of the isolates was amplified by Polymerase Chain Reaction (PCR) using the primer pair; ITS4 and ITS5. Results: The sequences of the amplified ITS region were blasted against known sequences on the National Centre for Biotechnology Information (NCBI) database. Nucleotide sequence analysis revealed the species identity of the fungal isolates to be: Aspergillus fumigatus, Trichoderma harzianum, Aspergillus felis, Aspergillus templicola, Aspergillus flavipes, Aspergillus fumigatus and Cunninghamella binariae. Phylogenetic analysis was carried out to ascertain the relationship between the isolates and other closely-related isolates on GenBank. Isolates 2 (Trichoderma harzianum) and 7 (Cunninghamella binariae), 3 (Aspergillus felis) and 6 (Aspergillus fumigatus), and 4 (Aspergillus templicola) and 5 (Aspergillus flavipes) were found to be more closely related to each other. Conclusion: The molecular techniques employed successfully identified the organisms to the species level as these techniques are based on the genetic constitution of organisms. The result obtained from this study will complement the information on the fungal organisms associated with dumpsite soil.


2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Ítalo Thiago Silveira Rocha Matos ◽  
Vanderly Andrade de Souza ◽  
Giovana do Rosário D’Angelo ◽  
Spartaco Astolfi Filho ◽  
Edson Júnior do Carmo ◽  
...  

Considering the high biotechnological potential of yeasts associated to edible fruits, a screening for these microorganisms, capable of alcoholic fermentation, was performed in ripe fruits of camu-camu (Myrciaria dubia, Kunth). The fruits were collected from north of Brazilian Amazon, in the floodplain of the Cauamé River. Yeasts were isolated, and fermentation capability was evaluated using Durham tubes. Quantitative assays were performed to calculate ethanol yield (g g−1), specific growth rate (h−1), and ethanol productivity (g L−1·h−1). Taxonomic identification was performed by ribosomal gene nucleotide sequence analysis by alignment using BLASTN. A total of fifteen yeast colonies were isolated, and three of them presented promising ability to ferment glucose to ethanol. These isolates were identified as Candida orthopsilosis, Pichia kudriavzevii, and Meyerozyma caribbica. When cultured in broth containing 180 g·L−1 of glucose, M. caribbica CC003 reached 91.7 percent of the maximum theoretical ethanol concentration (84.4 g·L−1), presenting an ethanol yield and productivity of 0.4688 g·g−1 and 0.781 g·L−1·h−1, respectively. These results indicate a promising potential of this isolate for bioprocess applications. This paper is a rare report of C. orthopsilosis with endophytic habit because most of the references indicate it as a human pathogen. Besides this, M. caribbica is a promising fermenter for alcoholic beverages due to its osmotolerance and high ethanol yield. This is the first paper reporting endophytic yeasts associated with fruits of Myrciaria dubia.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Seinen Chow ◽  
Takashi Yanagimoto ◽  
Haruko Takeyama

AbstractPartial mtDNA cytochrome oxidase subunit I (COI) fragments and near entire stretch of 12S rDNA (12S) and control region (Dloop) of the Japanese spiny lobster (Panulirus japonicus) (n = 3) were amplified by PCR and used for direct nucleotide sequencing and for clone library-based nucleotide sequence analysis. Nucleotide sequences of a total of 75 clones in COI, 77 in 12S and 92 in Dloop were determined. Haplotypes of the clones matched with those obtained by direct sequencing were determined to be genuine mtDNA sequence of the individual. Phylogenetic analysis revealed several distinct groups of haplotypes in all three regions. Genuine mtDNA sequences were observed to form a group with their closely related variables, and most of these variables may be due to amplification error but a few to be heteroplasmy. Haplotypes determined as nuclear mitochondrial pseudogenes (NUMTs) formed distinct groups. Nucleotide sequence divergence (K2P distance) between genuine haplotypes and NUMTs were substantial (7.169–23.880% for COI, 1.336–23.434% for 12S, and 7.897–71.862% for Dloop). These values were comparable to or smaller than those between species of the genus Panulirus, indicating that integration of mtDNA into the nuclear genome is a continuous and dynamic process throughout pre- and post-speciation events. Double peaks in electropherograms obtained by direct nucleotide sequencing were attributed to common nucleotides shared by multiple NUMTs. Information on the heteroplasmy and NUMTs would be very important for addressing their impact on direct nucleotide sequencing and for quality control of nucleotide sequences obtained.


2021 ◽  
Vol 26 (5) ◽  
pp. 8-15
Author(s):  
Ghasaq Albrqawy ◽  
A.S.Saadon

This study was conducted in the Laboratory of Fungus in the Department of Biology / College of Science / University of Qadisiyah to isolate and diagnose some insulation from fungi isolated from imported dried fruits (raisins) in Qadisiyah province, Iraq. The isolations were diagnosed both morphologically and microscopically using the classification keys and to confirm the appearance and microscopic diagnosis diagnosed using polymerase chain reaction(PCR), And determine the sequence of nitrogen bases (Nucleotide sequence(of compound DNA products using ITS1 and ITS4. The results of the nucleotide sequence analysis of DNA (PCR product) compounding innate isolation and using BLAST to compare with data available at the U.S. National Center for Biotechnology Information (NCBI) have shown that this isolation belongs to the type Monascus purpureus. By comparing the sequence of nitrogen bases of isolated M. purpureus fungus in this study, it was found that there was a 100% similarity to many of the M. purpureus fungus isolates previously registered at the National Center for Biotechnology Information (NCBI), including those diagnosed in China (MT361825, MK359689, MW581230) and Japan (AB477248).


2021 ◽  
Vol 31 (1) ◽  
Author(s):  
Kamal A. M. Abo-Elyousr ◽  
Sabry A. Hassan

Abstract Background Bacterial wilt of tomato (BWP) caused by Ralstonia solanacearum (Smith) is a very important disease. Biological control of this disease is a very important tool to protect the plant and environment from pollution of chemical control. Results Twenty isolates of genus, Pantoea were isolated from healthy tomato root. Out of 20 isolates, 2 strains, PHYTPO1 and PHYTPO2, showed highly antagonistic property to control the growth of R. solanacearum in vitro conditions. They were identified as P. agglomerans by using 16S rRNA nucleotide sequence analysis. The 2 isolates were selected to study their effect (as cell suspension or culture filtrate) on the bacterial wilt under greenhouse conditions. PHYTPO1 inhibited maximum growth reduction of R. solanacearum and formed 2.5 cm2 of inhibition zone, followed by 1.2 cm2 in PHYTOPO2 under in vitro conditions. Treating with both isolates of P. agglomerans was significantly reduced disease severity of tomato wilt disease. The disease severity was reduced to 74.1 when treated as cell suspension, while when treated as culture filtrate, it reduced the disease severity up to 69.4 than infected control. Conclusion The strains of Pantoea can be used as an ecofriendly method to control of the most economic pathogen of tomato under greenhouse conditions. Further study is needed to find an appropriate formulation and approving application of these bacteria under field conditions.


2021 ◽  
Author(s):  
Aleksey Perchun ◽  
Dmitry Pavlov ◽  
Vladimir Melnikov ◽  
Aleksey Antonychev ◽  
Nikolay Zinyakov ◽  
...  

Abstract The paper presents genetic characteristics of two strains of the spring viraemia of carp virus strains Kirov/08 and Orenburg/14 isolated in the Kirov and Orenburg Oblasts of the Russian Federation, respectively. The nucleotide sequence analysis of the 516-bp fragment of Kirov/08 and Orenburg/14 G gene showed 9.5% difference. The phylogenetic analysis demonstrated a high level of homology of these strains to SVC viruses, isolated earlier in Russia, Ukraine and the Republic of Moldova.


Pathogens ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 901
Author(s):  
Lucyna Kirczuk ◽  
Mariusz Piotrowski ◽  
Anna Rymaszewska

Tick-borne pathogens are an important medical and veterinary issue worldwide. Environmental monitoring in relation to not only climate change but also globalization is currently essential. The present study aimed to detect tick-borne pathogens of the genera Anaplasma, Rickettsia and Francisella in Ixodes ricinus ticks collected from the natural environment, i.e., recreational areas and pastures used for livestock grazing. A total of 1619 specimens of I. ricinus were collected, including ticks of all life stages (adults, nymphs and larvae). The study was performed using the PCR technique. Diagnostic gene fragments msp2 for Anaplasma, gltA for Rickettsia and tul4 for Francisella were amplified. No Francisella spp. DNA was detected in I. ricinus. DNA of A. phagocytophilum was detected in 0.54% of ticks and Rickettsia spp. in 3.69%. Nucleotide sequence analysis revealed that only one species of Rickettsia, R. helvetica, was present in the studied tick population. The present results are a part of a large-scale analysis aimed at monitoring the level of tick infestation in Northwest Poland.


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