scholarly journals Methods for DNA quantification yield similar relative but different absolute values

2019 ◽  
pp. 25-31
Author(s):  
O. P. Balanovsky ◽  
ZhA Kagazezheva ◽  
M. V. Olkova
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Correlation Coefficients ◽  
Dna Quantification ◽  
Sample Storage ◽  
Excellent Correlation ◽  
The Real ◽  
Absolute Concentrations

DNA quantification is a routine yet important procedure that determines the efficacy of long-term sample storage and further manipulations with the sample. There are a few well-established methods for measuring DNA concentrations. However, it still not fully clear how concordant their results are. The aim of this work was to measure DNA concentrations in a set of samples using different quantification methods and to compare the obtained values. In 2 independent experiments, a total of 100 genomic DNA samples were analyzed using 3 different DNA quantification methods, including spectrophotometry (NanoDrop), fluorometry (Qubit) and real-time PCR (Quantifiler). The obtained relative concentrations demonstrated an excellent correlation (the correlation coefficients were as high as 0.98 to 0.99). However, the absolute concentrations showed a considerable variation and even a twofold difference. Spectrophotometry yielded the highest concentrations, whereas fluorometry yielded the lowest. The real-time PCR results were intermediate. The differences were more pronounced for the samples with low DNA concentrations. We recommend that such differences should be accounted for when estimating DNA concentrations using an arsenal of different quantification methods.

APPLICATION OF REAL-TIME PRC TECHNIQUE AND REVERSE DOT-BLOT FOR DETECTION THE HIGH RISK TYPES OF HUMAN PAPILLOMAVIRUS CAUSING CERVICAL CANCER

2017 ◽  
pp. 99-103
Author(s):  
Van Bao Thang Phan ◽  
Hoang Bach Nguyen ◽  
Van Thanh Nguyen ◽  
Thi Nhu Hoa Tran ◽  
Viet Quynh Tram Ngo
Keyword(s):  
Cervical Cancer ◽  
High Risk ◽  
Real Time ◽  
Real Time Pcr ◽  
Dot Blot ◽  
The Real ◽  
Dot Blot Assay ◽  
Hpv Types ◽  
High Risk Hpv ◽  
Reverse Dot Blot

Introduction: Infection with HPV is the main cause of cervical cancer. Determining HPV infection and the types of HPV plays an important role in diagnosis, treatment and prognosis of cervicitis/cervical cancer. Aims: Determining proportion of high-risk HPV types and the occurrence of coinfection with multiple HPV types. Methods: 177 women with cervicitis or abnormal Pap smear result were enrolled in the study. Performing the real-time PCR for detecting HPV and the reverse DOT-BLOT assay for determining type of HPV in cases of positive PCR. Results: 7 types of high-risk HPV was dectected, the majority of these types were HPV type 18 (74.6%) and HPV type 16 (37.6%); the proportion of infection with only one type of HPV was 30.4% and coinfection with multiple HPV types was higher (69.6%), the coinfected cases with 2 and 3 types were dominated (32.2% and 20.3%, respectively) and the coinfected cases with 4 and 5 types were rare. Conclusion: Use of the real-time PCR and reverse DOT-BLOT assay can determine the high-risk HPV types and the occurrence of coinfection with multiple HPV types. Key words: HPV type, Reverse DOT-BLOT, real-time PCR,PCR, cervical cancer

scholarly journals Real-Time PCR as an Alternative Technique for Detection of Dermatophytes in Cattle Herds

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1662
Author(s):  
Dominik Łagowski ◽  
Sebastian Gnat ◽  
Aneta Nowakiewicz ◽  
Aleksandra Trościańczyk
Keyword(s):  
Light Microscopy ◽  
Real Time ◽  
Real Time Pcr ◽  
Direct Analysis ◽  
Carrier Status ◽  
The Real ◽  
Detection Techniques ◽  
Direct Light ◽  
Microscopy Analysis ◽  
Cattle Herds

Dermatophytes are filamentous fungi with the ability to digest and grow on keratinized substrates. The ongoing improvements in fungal detection techniques give new scope for clinical implementations in laboratories and veterinary clinics, including the monitoring of the disease and carrier status. The technologically advanced methods for dermatophyte detection include molecular methods based on PCR. In this context, the aim of this study was to carry out tests on the occurrence of dermatophytes in cattle herds using qPCR methods and a comparative analysis with conventional methods. Each sample collected from ringworm cases and from asymptomatic cattle was divided into three parts and subjected to the real-time PCR technique, direct light microscopy analysis, and culture-based methods. The use of the real-time PCR technique with pan-dermatophyte primers detected the presence of dermatophytes in the sample with a 10.84% (45% vs. 34.17%) higher efficiency than direct analysis with light microscopy. Moreover, a dermatophyte culture was obtained from all samples with a positive qPCR result. In conclusion, it seems that this method can be used with success to detect dermatophytes and monitor cowsheds in ringworm cases and carriers in cattle.

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

Real-Time PCR Determination of Escherichia coli Genomic DNA Contamination in Plasmid Preparations

2002 ◽  
Vol 301 (1) ◽  
pp. 151-153 ◽  
Author(s):  
Adrián Vilalta ◽  
Vanessa Whitlow ◽  
Terrie Martin
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Dna Contamination

scholarly journals Performance Evaluation of the Real-Q Cytomegalovirus (CMV) Quantification Kit Using Two Real-Time PCR Systems for Quantifying CMV DNA in Whole Blood

2016 ◽  
Vol 36 (6) ◽  
pp. 603-606 ◽  
Author(s):  
Jong Eun Park ◽  
Ji-Youn Kim ◽  
Sun Ae Yun ◽  
Myoung-Keun Lee ◽  
Hee Jae Huh ◽  
...  
Keyword(s):  
Performance Evaluation ◽  
Real Time ◽  
Real Time Pcr ◽  
Whole Blood ◽  
The Real

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
V. M. BOHAYCHUK ◽  
G. E. GENSLER ◽  
M. E. McFALL ◽  
R. K. KING ◽  
D. G. RENTER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Conventional Culture ◽  
Food Animal ◽  
Highly Sensitive ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

scholarly journals Touchable 3D hierarchically structured polyaniline nanoweb for capture and detection of pathogenic bacteria

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Kyung Hoon Kim ◽  
MinHo Yang ◽  
Younseong Song ◽  
Chi Hyun Kim ◽  
Young Mee Jung ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Bacterial Pathogens ◽  
Physical Contact ◽  
Structural Deformation ◽  
Pcr Analysis ◽  
Mechanical Resistance ◽  
Fluorescence Signal ◽  
The Real

AbstractA bacteria-capturing platform is a critical function of accurate, quantitative, and sensitive identification of bacterial pathogens for potential usage in the detection of foodborne diseases. Despite the development of various nanostructures and their surface chemical modification strategies, relative to the principal physical contact propagation of bacterial infections, mechanically robust and nanostructured platforms that are available to capture bacteria remain a significant problem. Here, a three-dimensional (3D) hierarchically structured polyaniline nanoweb film is developed for the efficient capture of bacterial pathogens by hand-touching. This unique nanostructure ensures sufficient mechanical resistance when exposed to compression and shear forces and facilitates the 3D interfacial interactions between bacterial extracellular organelles and polyaniline surfaces. The bacterial pathogens (Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus) are efficiently captured through finger-touching, as verified by the polymerase chain reaction (PCR) analysis. Moreover, the real-time PCR results of finger-touched cells on a 3D nanoweb film show a highly sensitive detection of bacteria, which is similar to those of the real-time PCR using cultured cells without the capturing step without any interfering of fluorescence signal and structural deformation during thermal cycling. Graphic Abstract

scholarly journals Susceptibility of Maize Inbreds and Incidence of Symptomless Infection by the Head Smut Pathogen, Sphacelotheca reiliana

2016 ◽  
Vol 17 (1) ◽  
pp. 1-5 ◽  
Author(s):  
S. J. Anderson ◽  
H. E. Simmons ◽  
R. D. French-Monar ◽  
G. P. Munkvold
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Field Trials ◽  
Growth Stages ◽  
High Susceptibility ◽  
Head Smut ◽  
Pcr Assay ◽  
Disease Symptoms ◽  
The Real ◽  
Seedling Infection

A real-time PCR assay was used to compare seedling infection by Sphacelotheca reiliana, the causal agent of head smut, among five inbred genotypes representing low, moderate, and high susceptibility to the disease. Seeds were coated with teliospores and planted in autoclaved field soil in a growth chamber. Incidence of seedling infection at growth stage V3 differed between an inbred genotype of low susceptibility and those of moderate and high susceptibility, but did not differ between the high and moderately susceptible groups (P < 0.05). The real-time PCR assay was also used to compare infection status at early and late vegetative stages with observable symptoms in the field. We detected infection via real-time PCR in maize at both growth stages during field trials conducted in Texas and California but observed no disease symptoms (smutted ears or tassels). Notably, the fungus was present in up to 31% of the ear shoots in plots without disease symptoms. The real-time assay can be a useful tool for screening seedling-stage host resistance, and for better understanding the progress of infection in different maize genotypes. The field data suggest that asymptomatic infection is much more common than previously thought, and may have important implications for the epidemiology of this fungus under diverse plant resistance and growing conditions. Accepted for publication 11 December 2015. Published 5 January 2016.

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