scholarly journals Analysis of the Results of DNA Isolation from Chicken Feather Sampled from the Base of the Young Feathers, the base of the Old Feathers and the Ends of the Feathers

BIOEDUSCIENCE ◽  
2021 ◽  
Vol 5 (2) ◽  
Author(s):  
Alfi Sophian
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Isolation ◽  
Average Concentration ◽  
Chicken Feathers ◽  
Average Value ◽  
Curve Method ◽  
Young Chicken ◽  
Negative Controls ◽  
Positive Controls

Background: The Microbiology and Molecular Biology testing laboratory at the Food and Drug Supervisory Agency in Gorontalo analyzed the results of DNA isolation from chicken feathers obtained from the base of young feathers, the base of elderly feathers, and the tips of the feathers. The goal was to provide information on the use of DNA templates in chicken samples so that the molecular research sampling process may employ feathers instead of hurting the test animals. The sample used consisted of 10 Bangkok chickens which were sampled for young feathers and old feathers and the tips of the feathers. Method: Quantitative techniques by comparing the results of DNA isolation which were analyzed using a nano photometer and then confirmed using real-time PCR with the SYBR green method. Result: The analysis of purity and concentration showed that at the base of young chicken feathers, the average value of purity was at 1,790, with an average value of the concentration of 4,210. At the base of the old feather, the average value of purity was 0.638, with an average concentration value that was not detected. Likewise, at the tip of the feather, the average purity value is 0.894 and the concentration value is not detected. Confirmation tests performed on all samples using the real-time PCR melt curve method showed that all samples were detected with a Tm value of 78.5 for young feathers, 78.5 for old feathers, 79.0 for positive controls and 78.7 for positive controls, while negative controls were not detected. Conclusion: DNA isolation can be carried out at the base of the young feathers, the base of the old feathers and the tips of the feathers.

scholarly journals Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1731
Author(s):  
Arianna Ceruti ◽  
Rea Maja Kobialka ◽  
Judah Ssekitoleko ◽  
Julius Boniface Okuni ◽  
Sandra Blome ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Isolation ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Recombinase Polymerase Amplification ◽  
Reference Laboratory ◽  
Analytical Sensitivity ◽  
Lysis Buffer

African swine fever virus (ASFV) is the causative agent of a deadly disease in pigs and is spread rapidly across borders. Samples collected from suspected cases must be sent to the reference laboratory for diagnosis using polymerase chain reaction (PCR). In this study, we aimed to develop a simple DNA isolation step and real-time recombinase polymerase amplification (RPA) assay for rapid detection of ASFV. RPA assay based on the p72 encoding B646L gene of ASFV was established. The assays limit of detection and cross-reactivity were investigated. Diagnostic performance was examined using 73 blood and serum samples. Two extraction approaches were tested: silica-column-based extraction method and simple non-purification DNA isolation (lysis buffer and heating, 70 °C for 20 min). All results were compared with well-established real-time PCR. In a field deployment during a disease outbreak event in Uganda, 20 whole blood samples were tested. The assay’s analytical sensitivity was 3.5 DNA copies of molecular standard per µL as determined by probit analysis on eight independent assay runs. The ASFV RPA assay only detected ASFV genotypes. Compared to real-time PCR, RPA diagnostic sensitivity and specificity were 100%. Using the heating/lysis buffer extraction procedure, ASFV-RPA revealed better tolerance to inhibitors than real-time PCR (97% and 38% positivity rate, respectively). In Uganda, infected animals were identified before the appearance of fever. The ASFV-RPA assay is shown to be as sensitive and specific as real-time PCR. Moreover, the combination of the simple extraction protocol allows its use at the point of need to improve control measures.

scholarly journals PRIMARY SLX TEST USING REAL-TIME PCR BASED ON HIGH RESOLUTION MELTING (HRM) ON MICROFILARIA EXAMINATION

2021 ◽  
Vol 5 (1) ◽  
pp. 26
Author(s):  
Bagus Muhammad Ihsan ◽  
Cecep Dani Sucipto ◽  
Khayan Khayan
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Wuchereria Bancrofti ◽  
Positive Control ◽  
Amplification Curve ◽  
Ct Value ◽  
Polymerase Chain ◽  
Positive Controls

Background: Filariasis patients can be a source of transmission if their blood still contains microfilariae. One of the Polymerase Chain Reaction (PCR) methods used is High Resolution Melting (HRM), using primary specificity testing. Purpose: To test the specificity of SLX primer. The samples used for this test were isolates of Salmonella., Klebsiella, Pseudomonas, negative and positive controls for Brugia malayi and Wuchereria bancrofti. Method: The design in this study is a quasi-experiment by testing the specificity of SLX primer using HRMbased real-time PCR based on the Cycle Threshold (CT) value observed through the amplification curve. Result: The real-time PCR results showed that no CT was released in the bacterial samples, and there was a CT value in the positive control. The results of this study indicate that specific SLX primer can be used in identifying microfilariae. Conclusion: SLX primer have a reasonable specificity because they cannot detect the existence of microorganisms in the samples other than microfilariae.

Synthesis of O-serogroup specific positive controls and real-time PCR standards for nine clinically relevant non-O157 STECs

2012 ◽  
Vol 91 (1) ◽  
pp. 52-56 ◽  
Author(s):  
Cheyenne C. Conrad ◽  
Brandon H. Gilroyed ◽  
Tim A. McAllister ◽  
Tim Reuter
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Positive Controls

DETECTION OFGIARDIA LAMBLIA, CRYPTOSPORIDIUMSPP. ANDENTAMOEBA HISTOLYTICAIN CLINICAL STOOL SAMPLES BY USING MULTIPLEX REAL-TIME PCR AFTER AUTOMATED DNA ISOLATION

2013 ◽  
Vol 68 (3) ◽  
pp. 188-192 ◽  
Author(s):  
P Van Lint ◽  
JW Rossen ◽  
S Vermeiren ◽  
K Ver Elst ◽  
S Weekx ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Isolation ◽  
Stool Samples

Quantitation of HTLV-I proviral load by a real-time PCR assay using SYBR Green: Comparison of two methods for DNA isolation

2010 ◽  
Vol 170 (1-2) ◽  
pp. 160-164 ◽  
Author(s):  
Natalia Andrea Altamirano ◽  
Carlos Rocco ◽  
Paula Aulicino ◽  
Luisa Sen ◽  
Andrea Mangano
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Proviral Load ◽  
Dna Isolation ◽  
Sybr Green ◽  
Pcr Assay

scholarly journals Comparison of Methods for DNA Isolation from Food Samples for Detection of Shiga Toxin-Producing Escherichia coli by Real-Time PCR

2003 ◽  
Vol 69 (3) ◽  
pp. 1844-1846 ◽  
Author(s):  
Loree C. Heller ◽  
Carisa R. Davis ◽  
K. Kealy Peak ◽  
David Wingfield ◽  
Andrew C. Cannons ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Dna Isolation ◽  
Food Samples ◽  
Pcr Detection ◽  
Comparison Of Methods ◽  
Commercial Kits ◽  
Shiga Toxin Genes

ABSTRACT In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.

scholarly journals Single gene targeted nanopore sequencing enables simultaneous identification and antimicrobial resistance detection of sexually transmitted infections

2020 ◽  
Author(s):  
Liqing Zhou ◽  
Andrea Lopez Rodas ◽  
Luz Marina Llangarí ◽  
Natalia Romero Sandoval ◽  
Philip Cooper ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Sexually Transmitted Infections ◽  
Real Time ◽  
Real Time Pcr ◽  
Single Gene ◽  
Vaginal Swab ◽  
Nanopore Sequencing ◽  
Sexually Transmitted ◽  
Negative Controls ◽  
Simultaneous Identification

AbstractObjectivesTo develop a simple DNA sequencing test for simultaneous identification and antimicrobial resistance (AMR) detection of multiple sexually transmitted infections (STIs).MethodsA total of 200 vulvo-vaginal swab samples from 200 female sex workers in Ecuador were initially tested by real-time PCR for Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV). Samples positive for these STIs and controls were subjected to single gene targeted PCR MinION-nanopore sequencing by using the smartphone operated MinIT.ResultsAmong 200 vulvo-vaginal swab samples 43 were positive for at least one of the STIs by real-time PCR. Single gene targeted nanopore sequencing generally yielded higher pathogen specific reads in positive samples than negative controls. Of the 26 CT, NG or MG infections identified by real-time PCR, 25 were clearly distinguishable from the negative controls using sequence read count. Discrimination of TV positives from controls was poorer as many had low pathogen loads (cycle threshold > 35) with associated low sequence read counts. AMR analysis workflow indicated that 11% of the classified reads aligned with antibiotic resistance genes, all of which identified fluoroquinolone resistance in NG.ConclusionsSingle gene targeted nanopore sequencing for diagnosing and simultaneously identifying key antimicrobial resistance markers for four common STIs, associated with genital tract discharge in women shows promise. Further work to optimise accuracy, reduce costs and improve speed may allow sustainable approaches for managing STIs and emerging AMR in resource poor and laboratory limited settings.

Sign in / Sign up

Export Citation Format

Share Document