Investigation of duck plague virus in hoar areas of Bangladesh

2020 ◽  
Vol 26 (1-2) ◽  
pp. 73-78
Author(s):  
A Hossen ◽  
MH Rahman ◽  
MZ Ali ◽  
MA Yousuf ◽  
MZ Hassan ◽  
...  
Keyword(s):  
Cytopathic Effect ◽  
Clinical Sample ◽  
Chorioallantoic Membrane ◽  
Clinical Samples ◽  
Isolation And Identification ◽  
Duck Plague Virus ◽  
Pcr Method ◽  
Polymerase Chain ◽  
The Individual ◽  
Free Ranging

Duck plague (DP) is the most important infectious disease of geese, ducks and free-ranging water birds. The present study was conducted to determine the prevalence of duck plague virus followed by isolation and identification. For these purposes, a total of 155 cloacal swabs samples were collected randomly from duck of different haor areas of Bangladesh including 45 (41 surveillance and 4 clinical) samples from Netrokona; 42 (40 surveillance and 2 clinical) samples from Kishoregonj; 30 samples from Brahmanbaria and 38 samples from Sunamganj. The samples were processed and pooled (1:5 ratio) for initial screening of target polymerase gene of duck plague virus by polymerase chain reaction (PCR) method. All the samples of a positive pool were then tested individually for identifying the individual positive samples. The result showed that out of 155 samples, 41 (26.45%) were found positive in which 17 were from Netrokona, where 15 (36.58%) were from surveillance samples and 2 (50%) were from clinical sample; 16 were from Kishoregonj, where 14 (35%) were from surveillance samples and 2 (100%) were from clinical sample; 2 (6.6%) were from Brahmanbaria and 5 (13.15%) were from Sunamganj. These positive samples were inoculated into 9-10 days embryonated duck eggs (EDE) through chorioallantoic membrane (CAM) route for the isolation of virus. The EDE died earlier was also chilled, and in a similar way, the CAMs were collected and again performed PCR for id entification of virus. Out of 41 PCR positive samples, 26 samples were isolated and reconfirmed by PCR. Subsequently, DPV was isolated in primary duck embryo fibroblasts cell culture and confirmed by observing cytopathic effect (CPE). Bang. J. Livs. Res. Vol. 26 (1&2), 2019: P. 73-78

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

scholarly journals Isolation and identification of Acanthamoeba genotypes and Naegleria spp. from the water samples of public swimming pools in Qazvin, Iran

2019 ◽  
Vol 18 (2) ◽  
pp. 244-251 ◽  
Author(s):  
Nastaran Paknejad ◽  
Elham Hajialilo ◽  
Mehrzad Saraei ◽  
Amir Javadi
Keyword(s):  
Water Samples ◽  
Polymerase Chain Reaction Amplification ◽  
Swimming Pools ◽  
Isolation And Identification ◽  
Highly Pathogenic ◽  
Reaction Amplification ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Naegleria Lovaniensis ◽  
Isolated Species

Abstract Free-living amoeba (FLA), including Acanthamoeba and Naegleria are facultative parasites in humans. The amoeba have widespread distribution in various water sources. The aim of this study was isolation and molecular identification of Acanthamoeba and Naegleria isolated from swimming pools and also hot and cold tub waters in Qazvin province. The samples (166 water samples) were cultured to isolate and identify positive specimens. PCR (polymerase chain reaction) amplification, sequencing and phylogenetic analysis were conducted to confirm the isolated species and genotypes of amoeba. According to morphological characterizations, 18.6% of specimens were identified as FLA, which in 71% were Acanthamoeba by PCR method. Molecular analysis revealed that 36.3%, 18.1% and 4.5% of Acanthamoeba specimens were identified as T3, T4 and T11 Acanthamoeba genotypes, respectively. Protacanthamoeba bohemica (27.2%) and Acanthamoeba sp. (4.5%) were found among the specimens. The results of osmo-tolerance and thermo-tolerance assays demonstrated that 50% of T3 and 25% of T4 genotypes of Acanthamoeba were highly pathogenic parasites. The molecular approach showed the presence of Naegleria lovaniensis (9%) in hot tub water of swimming pools. This study demonstrated that the swimming pools and hot tub water in Qazvin province were contaminated with Acanthamoeba and Naegleria species.

Malassezia japonica is part of the cutaneous microbiome of free-ranging golden-headed lion tamarins (Leontopithecus chrysomelas – Kuhl, 1820)

2019 ◽  
Vol 58 (1) ◽  
pp. 133-136
Author(s):  
Selene Dall’ Acqua Coutinho ◽  
Carlos Sacristán ◽  
Marina Galvão Bueno ◽  
Juliana Marigo ◽  
Alcides Pissinatti ◽  
...  
Keyword(s):  
Ribosomal Gene ◽  
Clinical Samples ◽  
External Ear ◽  
Chain Reaction ◽  
High Identity ◽  
Leontopithecus Chrysomelas ◽  
Lion Tamarins ◽  
Polymerase Chain ◽  
Malassezia Spp ◽  
Free Ranging

Abstract We investigated Malassezia spp. in external ear canal and haircoat of free-ranging golden-headed lion tamarins (Leontopithecus chrysomelas). A total of 199 animals were restrained, and 597 clinical samples were collected. After the amplification of the 26S ribosomal gene by polymerase chain reaction (PCR), the RFLP technique was performed. Two additional PCR protocols were performed in 10 randomly selected strains. Malassezia sp. was isolated in 38.2% (76/199) of the animals and 14.6% (87/597) of the samples; all strains were lipodependent. The 10 sequenced strains showed a high identity with Malassezia japonica, species described in man, but not in animals, so far.

scholarly journals Isolation of Infectious Bronchitis Virus in Primary cells of the Chick Embryo Chorioallantoic Membrane

2013 ◽  
Vol 37 (1) ◽  
pp. 109-114
Author(s):  
M. H. Mohammed
Keyword(s):  
Chick Embryo ◽  
Infectious Bronchitis Virus ◽  
Cytopathic Effect ◽  
Chorioallantoic Membrane ◽  
Rt Pcr ◽  
Infectious Bronchitis ◽  
Virus Titration ◽  
Chick Embryo Chorioallantoic Membrane ◽  
Polymerase Chain ◽  
Indirect Immunoperoxidase

The susceptibility of the primary chick embryo chorioallontoic membrane cells to infectious bronchitis virus was evaluated after twenty consecutive passages in chick embryo chorioallontoic membrane cells. Virus replication was monitored by cytopathic observation, indirect immunoperoxidase, and reverse transcription polymerase chain reaction (RT-PCR). At 72 hours post-infection in third passage, the cytopathic effect was characterized by rounding up of cells, monolayer detachment, intracytoplasmic brownish colouration was readily observed by immunoperoxidase from 24hours p.i in third passage, and at all times the extracted viral RNA from IBV-infected monolayers was demonstrated by RT-PCR. Tissue culture ineffective dose50 (TCID50) was used to measure virus titration performed on primary chick embryo chorioallontoic membrane cells and the titre in twenty passage was 108.6 TCID50/ml. The results obtained in this study suggested that the primary chick embryo chorioallontoic membrane cells can be used for adaptation infectious bronchitis virus (IBV) and may be considered a step forward for the use of these cells in the future for IBV vaccine production

scholarly journals Evaluation of two PCR-based procedures for typing Clostridium perfringens : research communication

2000 ◽  
Vol 71 (2) ◽  
Author(s):  
B. Dungu ◽  
M.M. Henton ◽  
A. Bosman ◽  
H. Fourie ◽  
G. Viljoen
Keyword(s):  
Multiplex Pcr ◽  
Clostridium Perfringens ◽  
Intradermal Test ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Skin Reactions ◽  
Large Numbers ◽  
Polymerase Chain ◽  
The Individual

Two polymerase chain reaction (PCR)-based procedures for typing Clostridium perfringens, which affects most domestic animals, were compared and evaluated for efficiency as substitute to the guinea-pig intradermal test routinely used in our laboratory, namely a multiplex PCR and a protocol based on the individual amplification of gene sequences specific for each toxin. Reference isolates of C. perfringens types A, B, C and D as well as cultures from clinical specimens were tested. The sensitivity and specificity of the PCR was confirmed on reference isolates. There was similarity in results on 43 of the 46 samples typed by all 3 methods. Clear results were obtained by PCR on 5 clinical samples that showed either equivocal or weak skin reactions in guinea-pigs. The multiplex PCR protocol, in combination with the evaluation of bacterial growth, is a better alternative to in vivo toxin typing, since C. perfringens can only be incriminated as cause of a disease when it is present in large numbers in the intestine.

scholarly journals Comparison of a novel real-time PCR method (RTA) and Artus RG for the quantification of HBV DNA and HCV RNA

2017 ◽  
Vol 11 (07) ◽  
pp. 543-548
Author(s):  
Sibel Aydogan ◽  
Ziya Cibal Acikgoz ◽  
Aysegul Gozalan ◽  
Fisun Kirca ◽  
Tuba Muderris ◽  
...  
Keyword(s):  
Quality Control ◽  
Real Time ◽  
Hbv Dna ◽  
Clinical Samples ◽  
Hcv Rna ◽  
Rt Pcr ◽  
B Virus ◽  
Pcr Method ◽  
Polymerase Chain

Introduction: The purpose of this study was to evaluate and compare two manual isolation and real-time (RT) polymerase chain reaction (PCR) kits (RTA RT-PCR with RTA isolation kit and Artus RG RT-PCR with QIAamp isolation kit) for molecular diagnosis of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Methodology: The study was conducted on 121 and 54 clinical samples for the detection of HBV DNA and HCV RNA, respectively, with an additional 8 HCV RNA external quality control samples. Results: Though a high correlation was observed between the two kits for the HBV DNA (r = 0.955, p = 0.001) and HCV RNA quantifications (r = 0.828, p = 0.001), discordant results were found in nine of the HBV DNA and in six of the HCV RNA samples. The mean difference between the two systems was found to be 0.4 log IU/mL in Quality Control for Molecular Diagnostics (QCMD) HCV RNA samples by Bland-Altman analysis. Conclusions: Although there was a high correlation between HBV DNA and HCV RNA tests according to the results of the study, the RTA system requires improvement for the determination of HCV RNA.

scholarly journals Isolamento e avaliação do comportamento do vírus ORF em células de córnea fetal caprina e identificação pela Reação em Cadeia da Polimerase

2021 ◽  
Vol 4 (4) ◽  
pp. 107-115
Author(s):  
Rosana Leo da Santana ◽  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cell Cultures ◽  
Cytopathic Effect ◽  
Clinical Symptoms ◽  
Small Variation ◽  
Clinical Samples ◽  
Vaccine Production ◽  
Chain Reaction ◽  
Cytoplasmic Vacuolization ◽  
Polymerase Chain

The control of the infection in endemic regions is performed with vaccines, however there are limitations in the vaccine production due to the difficulties in replicating the virus in cell cultures. This work was conducted so as to isolate and evaluate the behavior of ECV samples in fetal goat cell line cornea (CorFC) naturally immortalized. Crust samples from 22 sheep and seven goats presenting the clinical symptoms of EC from the states of Pernambuco, Bahia, Sergipe and Paraíba, were inoculated in CorFC monolayers and identified by polymerase chain reaction (PCR) using primers for amplification of a fragment of235 bp of gene B2L envelope ECV. Eleven samples were submitted to seven consecutive passes, at weekly intervals. Cytopathic effect (CPE) was observed in all passages, after24 hours post-infection, characterized by cell rounding, syncytial fusion with training, inclusion and cytoplasmic vacuolization. Thus, CorFC cell cultures proved highly permissible to ECV replication with small variation among viral samples. The PCR technique can be an efficient method used for confirmation of ECV infection in clinical samples.

scholarly journals Isolation and identification of Dichelobacter nodosus and Fusobacterium necrophorum using the polymerase chain reaction method in sheep with footrot

2015 ◽  
Vol 84 (2) ◽  
pp. 97-104 ◽  
Author(s):  
Ediz Kagan Ozgen ◽  
Seyda Cengiz ◽  
Mustafa Ulucan ◽  
Zafer Okumus ◽  
Asli Kortel ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Small Ruminants ◽  
Fusobacterium Necrophorum ◽  
Chain Reaction ◽  
Culture Methods ◽  
Isolation And Identification ◽  
Dichelobacter Nodosus ◽  
Specificity And Sensitivity ◽  
Pcr Method ◽  
Polymerase Chain

Footrot is an important infectious disease of small ruminants leading to severe economical losses. The aim of the present study was to determine isolation and identification rates ofDichelobacter nodosusandFusobacterium necrophorumin the culture techniques and reveal the specificity and sensitivity of the culture technique based on the polymerase chain reaction (PCR) method in sheep with footrot. Dry swabs and swabs with Amies medium from 83 sheep were subjected to PCR and culture analyses. In dry swabs, 4 samples were positive forF. necrophorumand all were negative forD. nodosus. Colonies in Eugon and Fusobacterium selective agars from swabs with Amies medium were evaluated. Polymerase chain reaction analysis was conducted on macroscopically and microscopically unidentified samples. The positivity rate was 55.4% forD. nodosusand 69.8% forF. necrophorumin cultures from Fusobacterium selective agars. The positivity rate forD. nodosusin Fusobacterium selective agars was higher than that in Eugon agar. Performing PCR and culture methods increased positivity as compared to performing them alone. In comparison with the PCR method, culturing in Fusobacterium selective agars had moderate sensitivity and low specificity forD. nodosus(71.7 and 28.7%) andF. necrophorum(61.3 and 80.0%), respectively. In conclusion, Fusobacterium selective agar (without antibiotics) for isolation and identification ofD. nodosusis superior to Eugon agar.Fusobacterium necrophorumshould also be considered as a provoking agent for footrot in small ruminants. The PCR method on culture increases elucidation of definitive aetiology.

scholarly journals EFFECTIVENESS OF MENIRAN (PHYLLANTHUS NIRURI LINN) AS ANTIBACTERIAL FOR RESISTANCE ANTIBIOTICS PREVENTION OF ENTEROTOXIN ESCHERICHIA COLI

2018 ◽  
Vol 7 (2) ◽  
pp. 35
Author(s):  
Sri Hidanah ◽  
Emy Koestanti Sabdoningrum ◽  
Retno Sri Wahyuni ◽  
Arini Rahmi Dewi ◽  
Erma - Safitri
Keyword(s):  
Escherichia Coli ◽  
The Body ◽  
Control Group ◽  
Anova Analysis ◽  
Chain Reaction ◽  
Isolation And Identification ◽  
Phyllanthus Niruri ◽  
E Coli ◽  
Pcr Method ◽  
Polymerase Chain

ABSTRACT               Escherichia coli (E. coli) can be isolated from the environment both inside and outside the hospes body. There were 89 serotypes in which 21% showed resistance to various antibiotics, such as E. coli enterotoxin. Alternative efforts were needed as a substitute for antibiotics, one of them through the use of medicinal plants, such as meniran (Phyllanthus niruri Linn).  Meniran plant is an immunomodulator that serves to repair the immune system of the body. The research was done through several stages: isolation and identification of  E. coli enterotoxin from several broiler farms in East Java using the polymerase chain reaction (PCR) method, E. coli resistance test against some antibiotics, making meniran extract and activation test against E. coli enterotoxin The study was divided into five treatments: T0+ (group of chickens were infected by E. coli enterotoxin), T0- (control group, not infected), T1 (infected by E. coli enterotoksin + 20% meniran extract), T2 (infected by E. coli enterotoksin + 25% extract meniran), T3 (infected by E. coli enterotoxin + 30% extract meniran). Data were analyzed by ANOVA (Analysis of Variance). The results were showed that all of  E. coli DNA isolates which tested by the PCR method was showed positive reactions at 600 bp. In the next stage, that E. coli enterotoxin are resistance to some antibiotics, such as Amoxicillin, Amphicillin, Erythromycin, Cephalosporins, Tetracycline, Cloxacillin and Gentamicin. Furthermore, 30% Phyllanthus niruri linn extract  effective as an antibacterial for the prevention of antibiotic resistance from E. coli enterotoxin. 

scholarly journals Discontinuation of isolation for persons with COVID-19: Is 10 days really safe?

2021 ◽  
Author(s):  
Alvina Clara Felix ◽  
Anderson V de Paula ◽  
Andreia Cristina Ribeiro ◽  
Francini Camila da Silva ◽  
Marta Inemami ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cytopathic Effect ◽  
Surrogate Marker ◽  
Patient Discharge ◽  
Clinical Samples ◽  
Cell Culture System ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Direct Indication ◽  
Polymerase Chain

AbstractBackgroundThe detection of SARS-CoV-2 RNA by real-time polymerase chain reaction (PCR) in respiratory samples from COVID-19 patients is not a direct indication of the presence of viable viruses. The isolation of SARS-CoV-2 in cell culture system however, can acts as surrogate marker of infectiousness. Cell culture based studies performed mostly with hospitalized and moderate/severe COVID-19 claims that no replication competent virus is found after 9 days of the symptoms onset in respiratory samples. Therefore, it is now recommended 10 days isolation before patient discharge.MethodsWe cell-cultured 29 SARS-COV-2 RT-PCR positive respiratory samples at the 10th day after the illness in Vero E6 cells. After two passages, cytopathic effect and cycle threshold (CT) lower than the obtained in the original sample were used to determine positivity.FindingsWe found viable particles in (7/29) 24% of samples tested. The positivity in cell culture was strongly associated (p<0.0001) to the low cycle thresholds in clinical samples (Ct <21).ConclusionThis data adds important knowledge to the current protocols for de-isolation of patients with non-hospitalized mild COVID-19.

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