scholarly journals TaqMan quantitative real-time PCR for detecting Avipoxvirus DNA in various sample types from hummingbirds

2020 ◽  
Author(s):  
Hanna E. Baek ◽  
Ravinder N. Sehgal ◽  
Ruta R. Bandivadekar ◽  
Pranav Pandit ◽  
Michelle Mah ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Disease Ecology ◽  
Core Protein ◽  
Bird Species ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Wide Range ◽  
Avian Pox ◽  
The Impact

AbstractBackgroundAvian pox is a viral disease documented in a wide range of bird species. Disease related detrimental effects can cause dyspnea and dysphagia, therefore birds with high metabolic requirements, such as hummingbirds, are especially vulnerable. Hummingbirds have a strong presence in California, especially in urban environments; however, little is understood regarding the impact of pox virus on hummingbird populations. Diagnosing pox infections relies on obtaining a tissue biopsy that poses significant bird risks and field challenges. Understanding the ecology of hummingbird pox viral infections could be advanced by a minimally invasive ante-mortem diagnostic method. This study’s goal was to address this gap in understanding if pox infections can be diagnosed using integumentary system samples besides tissue biopsies. To meet this goal, we tested multiple integumentary sample types and tested them using a quantitative real-time PCR assay. A secondary study goal was to determine which sample types (ranging from minimally to highly invasive sampling) were optimal for identifying infected birds.Methodology/Principal FindingsLesion tissue, pectoral muscle, feathers, toenail, blood, and swabs (both lesion tissue and non-lesion tissues) were taken from live birds and carcasses of two species of hummingbirds found in California. To maximize successful diagnosis, especially for samples with low viral load, a real-time quantitative PCR assay was developed for detecting the hummingbird-specific Avipoxvirus 4b core protein gene. Avipoxvirus DNA was successfully amplified from all sample types across 27 individuals. Our results were then compared to those of conventional PCR. Comparisons were also made between sample types utilizing lesion tissue samples as the gold standard.Conclusions/SignificanceHummingbird avian pox can be diagnosed without relying on tissue biopsies. Feather samples can be used for diagnosing infected birds and reduces sampling risk. A real-time PCR assay detected viral DNA in various integumentary system sample types and could be used for studying hummingbird disease ecology in the future.

Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum)

2008 ◽  
Vol 375 (1) ◽  
pp. 150-152 ◽  
Author(s):  
Cheng Xin Yi ◽  
Jun Zhang ◽  
Ka Man Chan ◽  
Xiao Kun Liu ◽  
Yan Hong
Keyword(s):  
Gossypium Hirsutum ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Transgene Copy Number

scholarly journals Virological Diagnosis of Herpes Simplex Virus 1 Esophagitis by Quantitative Real-Time PCR Assay

2011 ◽  
Vol 50 (3) ◽  
pp. 948-952 ◽  
Author(s):  
J.-F. Jazeron ◽  
C. Barbe ◽  
E. Frobert ◽  
F. Renois ◽  
D. Talmud ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Herpes Simplex Virus 1 ◽  
Virological Diagnosis ◽  
Simplex Virus

scholarly journals A Simplified Quantitative Real-Time PCR Assay for Monitoring SARS-CoV-2 Growth in Cell Culture

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Christian Shema Mugisha ◽  
Hung R. Vuong ◽  
Maritza Puray-Chavez ◽  
Adam L. Bailey ◽  
Julie M. Fox ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Dna Viruses ◽  
Culture Supernatants ◽  
Hiv 1 ◽  
Rna And Dna

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.

scholarly journals Examination of Mycobacterium avium subsp. avium distribution in naturally infected hens by culture and triplex quantitative real time PCR

2010 ◽  
Vol 55 (No. 7) ◽  
pp. 325-330 ◽  
Author(s):  
M. Kaevska ◽  
I. Slana ◽  
P. Kralik ◽  
I. Pavlik
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Clinical Symptoms ◽  
Bird Species ◽  
Etiologic Agent ◽  
Mycobacterium Avium Subsp ◽  
Quantitative Real Time Pcr ◽  
Tissue Samples ◽  
Conventional Culture

Mycobacterium avium subsp. avium (MAA) is the etiologic agent of avian tuberculosis, a chronic contagious disease described in a wide variety of domestic and wild bird species. The aims of this study were to assess the advantages of triplex quantitative real time PCR (qPCR) in comparison with culture testing for distribution of MAA in the organs of hens displaying varying degrees of clinical symptoms of the disease. From one small flock of ten hens and one cock with a history of weight loss, 98 tissue samples were examined in total. Pathological lesions were observed in six hens from which two were clinically ill. A total of 12 samples were positive by culture and 16 were positive by IS901 and IS1245 qPCR, confirming MAA infection. In conclusion, qPCR was a faster and more reliable alternative method in comparison with conventional culture analysis. Due to the detection of MAA in the muscle tissue of one hen, consumption of under cooked meat originating from infected fowl could pose a threat to immunosuppressed individuals.

A Quantitative Real-time PCR Assay for Quantification of Viable Listeria Monocytogenes Cells After Bacteriocin Injury in Food-First Insights

2010 ◽  
Vol 61 (6) ◽  
pp. 515-519 ◽  
Author(s):  
Antonio Cobo Molinos ◽  
Hikmate Abriouel ◽  
Nabil Ben Omar ◽  
Magdalena Martinez-Canamero ◽  
Antonio Gálvez
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay

scholarly journals Correction for Hilbert et al., Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis

2016 ◽  
Vol 54 (7) ◽  
pp. 1930-1930 ◽  
Author(s):  
David W. Hilbert ◽  
William L. Smith ◽  
Sean G. Chadwick ◽  
Geoffrey Toner ◽  
Eli Mordechai ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Development And Validation
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