scholarly journals The First Detection of Equine Coronavirus in Adult Horses and Foals in Ireland

Viruses ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 946 ◽  
Author(s):  
Nemoto ◽  
Schofield ◽  
Cullinane
Keyword(s):  
Polymerase Chain Reaction ◽  
Phylogenetic Analysis ◽  
Phylogenetic Analyses ◽  
Clinical Samples ◽  
Enteric Disease ◽  
Coding Region ◽  
Chain Reaction ◽  
Diagnostic Laboratory ◽  
Polymerase Chain ◽  
Thoroughbred Foals

The objective of this study was to investigate the presence of equine coronavirus (ECoV) in clinical samples submitted to a diagnostic laboratory in Ireland. A total of 424 clinical samples were examined from equids with enteric disease in 24 Irish counties between 2011 and 2015. A real-time reverse transcription polymerase chain reaction was used to detect ECoV RNA. Nucleocapsid, spike and the region from the p4.7 to p12.7 genes of positive samples were sequenced, and sequence and phylogenetic analyses were conducted. Five samples (1.2%) collected in 2011 and 2013 tested positive for ECoV. Positive samples were collected from adult horses, Thoroughbred foals and a donkey foal. Sequence and/or phylogenetic analysis showed that nucleocapsid, spike and p12.7 genes were highly conserved and were closely related to ECoVs identified in other countries. In contrast, the region from p4.7 and the non-coding region following the p4.7 gene had deletions or insertions. The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the Irish viruses were distinguishable from those circulating in other countries. This is the first report of ECoV detected in both foals and adult horses in Ireland.

Using a Polymerase Chain Reaction as a Complementary Test to Improve the Detection of Dermatophyte Fungus in Nails

2014 ◽  
Vol 104 (3) ◽  
pp. 233-237 ◽  
Author(s):  
María José Iglesias Sánchez ◽  
Ana María Pérez Pico ◽  
Félix Marcos Tejedor ◽  
María Jesús Iglesias Sánchez ◽  
Raquel Mayordomo Acevedo
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Classical Method ◽  
Clinical Manifestations ◽  
Culture Method ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Traditional Procedure ◽  
Polymerase Chain

Background Dermatomycoses are a group of pathologic abnormalities frequently seen in clinical practice, and their prevalence has increased in recent decades. Diagnostic confirmation of mycotic infection in nails is essential because there are several pathologic conditions with similar clinical manifestations. The classical method for confirming the presence of fungus in nail is microbiological culture and the identification of morphological structures by microscopy. Methods We devised a nested polymerase chain reaction (PCR) that amplifies specific DNA sequences of dermatophyte fungus that is notably faster than the 3 to 4 weeks that the traditional procedure takes. We compared this new technique and the conventional plate culture method in 225 nail samples. The results were subjected to statistical analysis. Results We found concordance in 78.2% of the samples analyzed by the two methods and increased sensitivity when simultaneously using the two methods to analyze clinical samples. Now we can confirm the presence of dermatophyte fungus in most of the positive samples in just 24 hours, and we have to wait for the result of culture only in negative PCR cases. Conclusions Although this PCR cannot, at present, substitute for the traditional culture method in the detection of dermatophyte infection of the nails, it can be used as a complementary technique because its main advantage lies in the significant reduction of time used for diagnosis, in addition to higher sensitivity.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals UTILITY OF ANTIGEN DETECTION TEST AND POLYMERASE CHAIN REACTION IN THE DIFFERENTIATION OF TUBERCULOUS AND NON-TUBERCULOUS MYCOBACTERIA

2017 ◽  
Vol 10 (6) ◽  
pp. 289
Author(s):  
Yogita Singh ◽  
Raji Vasanth ◽  
Shrikala Baliga ◽  
Dhanashree B
Keyword(s):  
Polymerase Chain Reaction ◽  
False Negative ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Chain Reaction ◽  
College Hospital ◽  
Medical College ◽  
Negative Results ◽  
Polymerase Chain ◽  
Mycobacterium Spp

Objectives: Cultivation and identification of mycobacteria to species level remains difficult and time-consuming. Hence, easy and rapid diagnostic methods are necessary for the differentiation of Mycobacterium tuberculosis (MTB) from non-tuberculous mycobacteria (NTM). The present study aims to detect and differentiate MTB from NTM isolated from clinical samples by immunochromatographic test (ICT) and polymerase chain reaction (PCR). Methods: Over a period of 1 year, clinical samples (n=496) received from suspected cases of TB, at the Department of Microbiology, Kasturba Medical College Hospital, Mangalore were cultured to isolate Mycobacterium spp. Identification of all the isolates was done by conventional biochemical technique, ICT, and PCR. Results: Among the 496 samples processed, 49 (9.87%) were acid-fast bacilli smear positive and 59 (11.89%) samples showed the growth of Mycobacterium spp. Among these, 10 were rapid growers, 49 were slow-growing mycobacteria, out of which 30 were MTB as identified by conventional biochemical reaction. Out of 59 Mycobacterial isolates subjected to ICT for the detection of MPT 64 antigen, only 28 were identified as MTB. However, all the 30 isolates were correctly identified as MTB by PCR. Conclusion: Hence, PCR is essential for rapid differentiation of non-tuberculous Mycobacterium from MTB. False negative results seen with immunochromatographic MPT 64 antigen assay could be due to mutations within the mpt64 gene. Further studies are necessary to characterize these PCR-positive and immunochromatographic assay negative MTB isolates.

scholarly journals Discovery of Cocirculating Ross River Virus and Barmah Forest Virus At Wide Bay Military Training Area, Northeastern Australia

2019 ◽  
Vol 35 (3) ◽  
pp. 220-223
Author(s):  
Jo Kizu ◽  
Christina Neuman ◽  
Luke Le Grand ◽  
Wenjun Liu
Keyword(s):  
Polymerase Chain Reaction ◽  
Phylogenetic Analysis ◽  
Military Training ◽  
Ross River Virus ◽  
Chain Reaction ◽  
Culex Annulirostris ◽  
Australian Defence Force ◽  
Polymerase Chain ◽  
Human Infections ◽  
Training Area

ABSTRACT An arbovirus surveillance military exercise was conducted to assess the risk of Ross River virus (RRV) and Barmah Forest virus (BFV) in the Australian Defence Force (ADF) Wide Bay training area (WBTA), northeastern Australia, in April 2018. Of the 5,540 female mosquitoes collected, 3,702 were screened for RRV and BFV by quantitative reverse transcription–polymerase chain reaction in a field laboratory. One pool of Verrallina funerea was positive for RRV and 8 pools (7 pools of Aedes vigilax and 1 pool of Culex annulirostris) were positive for BFV. Phylogenetic analysis of the complete nucleotide sequence of the E2 protein subgrouped both RRV and BFV with viruses previously isolated from human infections, indicating the potential risk of RRV and BFV infection to ADF personnel while training in WBTA. This is the 1st time that both RRV and BFV have been detected in a military training area.

scholarly journals Deteksi Transgen (Glu-1Dx5) pada Populasi Padi (Oryza sativa L.) Putative Transgenik Kultivar Fatmawati

Agrikultura ◽  
2010 ◽  
Vol 21 (1) ◽  
Author(s):  
Nono Carsono ◽  
Sri Nurlianti ◽  
Inez Nur Indrayani ◽  
Ade Ismail ◽  
Tri Joko Santoso ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Oryza Sativa ◽  
Dna Polymerase ◽  
Genomic Dna ◽  
Oryza Sativa L ◽  
Dna Purification ◽  
Coding Region ◽  
Chain Reaction ◽  
Taq Dna Polymerase ◽  
Polymerase Chain

Transformasi gen Glu-1Dx5, pengendali utama karakter elastisitas dan daya mengembang adonan dari gandum, telah berhasil ditransfer ke dalam genom tanaman padi kultivar Fatmawati dengan menggunakan penembakan partikel, dengan tujuan untuk memperbaiki kualitas adonan tepung beras. Galur-galur harapan telah diperoleh, tetapi karena telah mengalami penyerbukan sendiri selama 1-2 generasi yang menyebabkan transgen mengalami segregasi, maka diperlukan upaya pendeteksian transgen pada populasi putative transgenik ini. Upaya ini dapat dilakukan, antara lain dengan menggunakan teknik Polymerase Chain Reaction (PCR) yang memungkinkan perbanyakan fragmen DNA yang spesifik (gen) secara cepat dalam jumlah banyak.  Percobaan ini bertujuan untuk mendapatkan tanaman padi transgenik yang memiliki gen Glu-1Dx5 pada dua generasi yang sedang bersegregasi. DNA genom dari 149 tanaman padi (generasi T1 sebanyak 14 tanaman, generasi T2 sebanyak 134 tanaman, dan satu tanaman non-transgenik) telah diekstraksi menggunakan Genomic DNA Purification Kit dari Fermentas. Plasmid pK+Dx5 digunakan sebagai positif kontrol, selain itu digunakan juga enzim Taq DNA polymerase dari Go Green Taq® Master Mix (Promega) dan 2 primer spesifik yang mengamplifikasi coding region dari Glu-1Dx5 (2,5 kb). Hasil percobaan menunjukkan, tanaman padi yang memiliki gen Glu-1Dx5 pada generasi T2-7 sebanyak 26 tanaman, T2-11 : 12 tanaman, T2-12 : 3 tanaman, T2-40 : 3 tanaman dan T2-45 : 5 tanaman. Seluruh tanaman generasi T1 tidak memiliki insert. Hasil ini menunjukkan bahwa gen Glu-1Dx5 sudah terintegrasi ke dalam genom tanaman padi kultivar Fatmawati dan diwariskan dari satu generasi ke generasi berikutnya.

scholarly journals Puces à ADN comme outil d'identification moléculaire pour Culicoides

2009 ◽  
Vol 62 (2-4) ◽  
pp. 144
Author(s):  
J.C. De Witte ◽  
I. Deblauwe ◽  
Gill De Deken ◽  
R. De Deken ◽  
M. Madder ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Internal Transcribed Spacer ◽  
Culicoides Species ◽  
Chain Reaction ◽  
Diagnostic Laboratory ◽  
Laboratory Equipment ◽  
Naked Eye ◽  
Culicoides Obsoletus ◽  
Polymerase Chain ◽  
Obsoletus Complex

A DNA microarray test based on internal transcribed spacer 1 (ITS1) genotype expression was developed to identify Culicoides species of Northwestern Europe belonging to the Culicoides Obsoletus complex. The assay was designed so as to allow interpretation by the naked eye. False positive and false nega­tive results were eliminated. The need for expensive laboratory equipment and reagents was kept as low as possible, making the technique affordable and feasible for any diagnostic laboratory with polymerase chain reaction (PCR) facilities. The microarray test could be validated through the three ringtests organised by Medreonet. Use of this microarray can improve monitoring adult and immature Culicoides species.

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.

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