A fast one-step reverse transcription and polymerase chain reaction (RT-PCR) amplification procedure providing highly specific complementary DNA from plant virus RNA

2000 ◽  
Vol 87 (1-2) ◽  
pp. 25-28 ◽  
Author(s):  
A. Sambade ◽  
S. Martı́n ◽  
A. Olmos ◽  
M.L. Garcı́a ◽  
M. Cambra ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Plant Virus ◽  
Pcr Amplification ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Complementary Dna ◽  
Virus Rna ◽  
One Step ◽  
Polymerase Chain ◽  
Amplification Procedure

scholarly journals Development of multiplex reverse transcription polymerase chain reaction (RT-PCR) for simultaneous detection of matrix, haemagglutinin and neuraminidase genes of H5N1 avian influenza virus

2012 ◽  
Vol 28 (2) ◽  
pp. 55-59 ◽  
Author(s):  
R. Mojumder ◽  
E. H. Chowdhury ◽  
R Parvin ◽  
J. A. Begum ◽  
M. Giasuddin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Influenza Virus ◽  
Avian Influenza ◽  
Matrix Protein ◽  
Simultaneous Detection ◽  
High Fidelity ◽  
Rt Pcr ◽  
Chain Reaction ◽  
One Step ◽  
Polymerase Chain

Influenza A virus, subtype H5N1 causes a fatal disease in domestic poultry and could spread directly from poultry to humans. The aim of this study was to develop a multiplex reverse transcription polymerase chain reaction (mRT-PCR) for simultaneous detection of Type A influenza virus-specific matrix protein (M) gene as well as H5 and N1 genes of highly pathogenic avian influenza (HPAI) viruses. Finnzymes Phusion-Flash High- Fidelity PCR Master Mix (Finnzymes Oy, Finland) and Qiagen one-step RT-PCR enzyme mix (Qiagen, Germany) were used in a one-step RT-PCR. RNA was extracted from two known positive samples using Qiagen RNA extraction kit. RT-PCR was carried out with a mixture of primers specific for the Type A influenza virus matrix protein (M), and H5 and N1 genes of H5N1 HPAI viruses in a single reaction system. The mRT-PCR cDNA products were visualized by gel electrophoresis. The mRT-PCR yielded fragments of 245 bp for M, 545 bp for H5 and 343 bp for N1 genes of HPAI virus, which were clearly distinguishable. The mRT-PCR using the Finnzymes Phusion-Flash High-Fidelity PCR Master Mix (Finnzymes Oy, Finland) with Qiagen one-step RT-PCR Enzyme Mix (Qiagen, Germany) required only one hour and 20 minutes. (Bangl. vet. 2011. Vol. 28, No. 2, 55 – 59)DOI: http://dx.doi.org/10.3329/bvet.v28i2.10653

Persistent Measles Virus Infection and Otosclerosis

2001 ◽  
Vol 110 (10) ◽  
pp. 897-903 ◽  
Author(s):  
Wolfgang Arnold ◽  
Hans P. Niedermeyer ◽  
Maria Schuster ◽  
Wolfgang J. Neubert ◽  
Christa Baumann ◽  
...  
Keyword(s):  
Cell Culture ◽  
Polymerase Chain Reaction ◽  
Measles Virus ◽  
Otic Capsule ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Virus Rna ◽  
Polymerase Chain ◽  
H Protein ◽  
Antibody Pattern

The cause of otosclerosis is still unknown. Recently, measles virus involvement has been implicated. The aim of this study was to analyze the presence of measles virus RNA within the otosclerotic focus and to evaluate the perilymphatic antibody pattern. Bone and perilymph specimens from 40 patients with the spontaneous form of otosclerosis and from control patients were investigated by reverse transcription polymerase chain reaction (RT-PCR), Western blot techniques, and cell culture. By the use of RT-PCR, measles virus RNA could be detected in 32 patients, but not in controls. Analysis of perilymph revealed the presence of antibodies to N, F1, and M measles virus proteins in all cases, and antibodies against H protein in 2 additional cases. In preosteoblasts cultured from otosclerotic bone chips, no measles virus RNA could be amplified. We conclude that the spontaneous form of otosclerosis is, in the vast majority of cases, a measles virus-associated disease of the otic capsule.

scholarly journals A One-Step Reverse Transcription-Polymerase Chain Reaction System for the Simultaneous Detection and Identification of Multiple Tospovirus Infections

2005 ◽  
Vol 95 (2) ◽  
pp. 166-171 ◽  
Author(s):  
Hiroyuki Uga ◽  
Shinya Tsuda
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Simultaneous Detection ◽  
Yellow Spot ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Spot Virus ◽  
Detection And Identification ◽  
One Step ◽  
Polymerase Chain

A one-step reverse transcription-polymerase chain reaction (RT-PCR) method has been developed for the simultaneous detection and identification of multiple tospoviruses that infect plants. The RT-PCR system is composed of six primers in a single tube: a universal degenerate primer and five virus species-specific primers. Amplifications resulted in an 848-bp PCR product for Watermelon silver mottle virus, 709-bp for Tomato spotted wilt virus, 589-bp for Impatiens necrotic spot virus, 511-bp for Melon yellow spot virus, and a 459-bp amplicon for Iris yellow spot virus. This system enables the simultaneous detection of at least three types of tospovirus infections, in addition to their species identities, from five possible tospoviruses studied, on the basis of their S RNA combinations. This multiplex RT-PCR system was applied to the detection of tospovirus in ornamental crops cultivated in fields and shows potential for epidemiological studies.

scholarly journals Mosquito-specific viruses (family Flaviviridae, genus Flavivirus) Diisolasi pada Nyamuk Anopheles vagus di Bali

2021 ◽  
Vol 22 (2) ◽  
pp. 189-197
Author(s):  
Putu Ayu Asri Damayanti ◽  
I Nyoman Mantik Astawa ◽  
Anak Agung Ayu Mirah Adi ◽  
I Made Sudarmaja ◽  
I Kadek Swastika ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Light Trap ◽  
Baby Hamster Kidney ◽  
Rt Pcr ◽  
Chain Reaction ◽  
One Step ◽  
Polymerase Chain ◽  
Anopheles Vagus

Mosquito-specific viruses (MSVs) adalah virus yang hanya dapat bereplikasi pada sel nyamuk. Virus ini terdiri dari berbagai genus, salah satunya yang paling banyak ditemukan adalah dari famili Flaviviridae, genus Flavivirus. Namun, data keberadaan dan karakteristik MSVs dan vektornya di Bali saat ini sangat terbatas. Oleh karena itu, pengamatan untuk memperluas penemuan keragaman vektor dan filogenetik MSVs famili Flaviviridae, genus Flavivirus di Bali dilakukan pada tahun 2016-2018. Nyamuk-nyamuk dewasa ditangkap menggunakan light trap dan dikelompokkan berdasarkan spesies. Isolasi dan propagasi virus dilakukan pada galur sel C6/36 dan baby hamster kidney-21 (BHK-21). Identifikasi virus dilakukan dengan menggunakan one step reverse-transcriptase polymerase chain reaction (RT-PCR). Terdapat dua pool yang berasal dari nyamuk Anopheles vagus menampakan cythopathic effect (CPE) hanya pada galur sel C6/36 dari total 158 pool. Virus yang diisolasi memiliki persentase identity sekuen nukleotida tertinggi 97% dan sekuen asam amino 96% dengan virus Culex theileri Flavivirus isolat JKT-8650 yang diisolasi pada tahun 1981. Selanjutnya, virus dinamakan Mosquito Flavivirus Isolate Bali (MFB) dengan accession numbers KY995166 dan KY290258. Analisis filogenetik menunjukan bahwa MFB berada satu kluster dengan Culex theileri Flavivirus (CTFV) dari Indonesia, Culex Flavivuruses-Myanmar, Culex theileri Flavivirus-Portugal, dan Mosquito Flavivirus-Turki. Terdapat delapan nukelotida dan enam asam amino yang berbeda antara MFB dan CTFV Indonesia. Pada penelitian ini dapat disimpulkan bahwa MSVs dari famili Flaviviridae, genus Flavivirus berhasil diisolasi dari nyamuk An. vagus di Bali.

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