Comparison of three commercial DNA extraction kits for the enhancement of PCR assay sensitivity for Xanthomonas euvesicatoria pv. allii

2021 ◽  
Author(s):  
Nyasha J. Kavhiza ◽  
Meisam Zargar ◽  
Svetlana I. Prikhodko ◽  
Elena N. Pakina
Keyword(s):  
Dna Extraction ◽  
Pcr Assay ◽  
Assay Sensitivity ◽  
Xanthomonas Euvesicatoria

scholarly journals Determining the optimal method for DNA isolation from fruit jams

2018 ◽  
Vol 36 (No. 2) ◽  
pp. 126-132
Author(s):  
Sovová Tereza ◽  
Křížová Barbora ◽  
Ovesná Jaroslava
Keyword(s):  
Dna Extraction ◽  
Dna Isolation ◽  
Extraction Methods ◽  
Food Samples ◽  
Pcr Analysis ◽  
Uv Spectrophotometry ◽  
Pcr Assay ◽  
Optimal Method ◽  
Ctab Method ◽  
Dna Extraction Methods

DNA extraction is a crucial step in PCR analysis especially when analysing food samples that can be degraded and can potentially contain PCR-inhibiting substances. In this study, we compared the suitability of three DNA extraction methods – two kits: DNeasy<sup>®</sup> Plant Mini Kit and NucleoSpin<sup>®</sup> Food, and the CTAB method – for DNA extraction from commercial fruit jams. Fourteen jams with different contents of fruit, sugar and other additives were extracted in triplicate using the above-mentioned methods directly and after a washing step. The concentration and optical density were analysed using UV spectrophotometry and the amplifiability of the obtained DNA was evaluated using a PCR assay targeting a sequence coding for chloroplast tRNA-Leu. Samples isolated using the NucleoSpin<sup>®</sup> Food kit contained non-amplifiable DNA in eight cases, and samples isolated using the CTAB method could not be quantified. The DNeasy<sup>®</sup> Plant Mini Kit thus proved to be the most suitable method, since well-amplifiable DNA was obtained for all the analysed samples.

scholarly journals Comparason of extraction methods for PCR detection of Burkholderia cepacia complex (BCC) from cystic fibrosis patients

Open Medicine ◽  
2008 ◽  
Vol 3 (2) ◽  
pp. 157-162
Author(s):  
Koray Ergunay ◽  
Pinar Yurdakul ◽  
Burcin Sener ◽  
Ugur Ozcelik ◽  
Erdem Karabulut ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Burkholderia Cepacia ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Extraction Methods ◽  
Rapid Identification ◽  
Burkholderia Cepacia Complex ◽  
Assay Sensitivity ◽  
Spin Column ◽  
Significant Difference

AbstractDirect detection of Burkholderia cepacia complex (BCC) and its genomovars from sputum by molecular tests emerges as a method for rapid identification. In this study, four DNA extraction methods were evaluated for the identification for BCC from sputum of CF patients. Sputa from 28 CF patients were aliquoted and spiked with BCC reference strain. Boiling, phenol-chloroform, CTAB methods and a commercial spin column kit was used for DNA extraction. Total DNA yields were determined by spectrophotometry and single-round recA PCR was used for detection of BCC. No significant difference was observed in DNA yields from different extraction methods. Lower limit of detection for recA PCR was determined as 106 cfu/ml. Amplification was observed in 7/16 (43.7%) of sputa for boiling, 8/16 (50%) of sputa for CTAB and 13/16 (81.2%) of sputa for phenol-chloroform method and spin column kit in the assay sensitivity range determined in the study. Phenol-chloroform and commercial spin column kit were found to be better suited for DNA purification from sputum of CF patients for BCC identification. Diagnostic impact of single-round recA PCR directly from sputum was limited to chronically-infected patients.

Congenital cytomegalovirus infection management; what is new?

2021 ◽  
Vol 1 (2) ◽  
pp. 001-007
Author(s):  
Therese-Mary William
Keyword(s):  
Pregnant Women ◽  
Screening Program ◽  
Dried Blood Spots ◽  
Cmv Infection ◽  
Pcr Assay ◽  
Assay Sensitivity ◽  
Congenital Cytomegalovirus ◽  
Congenital Cmv Infection ◽  
Specific Symptoms ◽  
Congenital Cmv

Congenital Cytomegalovirus (CMV) infection is one of major public health concerns and one of the most frequent congenital infections worldwide. Congenital CMV infection is under-diagnosed in the majority of asymptomatic pregnant women due to its self-limited non-specific symptoms and unimplemented screening program. It also can go undetected at birth because in affected newborns are often asymptomatic; however, they manifest serious morbidities later in life. Primary CMV infections are associated with the highest in-utero transmission with estimated rates of 30–35% . Transmission occurs less frequently in secondary maternal infections at approximately 1.1–1.7%. There are growing evidences that early treatment with the antiviral therapy in newborns with congenital CMV infection can reduce sensorineural hearing loss (SNHL) and the substantial long-term neurodevelopmental disabilities. There is increased interest in establishing a prophylactic CMV vaccines that can protect seronegative mothers from primary infection and also to augment the immune response in seropositive women in order to prevent reactivation or re-infection. Saliva PCR assay can be used to screen newborns within the first 3 weeks of age instead of Dried blood spots (DBS), liquid-saliva PCR assay sensitivity is “100%” higher than DBS sensitivity “35%-100%" as not all infants have detectable viraemia at birth. Suggestive strategies to reduce the burden of congenital CMV disease and ensure better outcomes are; establishing a screening programme for pregnant mothers, developing prophylactic CMV vaccines, early therapeutic intervention in pregnant women and newborns and to replace DBS with Saliva PCR assay in screening newborns.

Distinguishing Bacteriolytic from Bacteriostatic Activity of an Antibiotic Agent by Real-Time Quantitative PCR

2011 ◽  
Vol 343-344 ◽  
pp. 357-360
Author(s):  
Zhi Ping Wang
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Bacteriostatic Activity ◽  
Antibiotic Agent ◽  
Pcr Assay ◽  
Assay Sensitivity ◽  
Real Time Quantitative Pcr ◽  
Pcr Product ◽  
Dna Template ◽  
Quantitative Pcr Assay

A simple and rapid real-time quantitative PCR assay was devised to discriminate bacteriolytic from bacteriostatic activity for a given antibacterial agent. Bacteria suspension was incubated with the compound solution, the mixture was centrifuged and supernatant was removed completely, the obtained pellet was then used as the DNA template for PCR. Then the bacteriostatic and bacteriolytic activity can be inferred by the quantity of PCR product. Moreover, the parameters that influence the assay sensitivity was discussed.

Evaluation of Three Real-Time PCR Methods for Detection of Salmonella from Cloves

2017 ◽  
Vol 80 (6) ◽  
pp. 982-989 ◽  
Author(s):  
Aparna Tatavarthy ◽  
Laila Ali ◽  
Vikas Gill ◽  
Lijun Hu ◽  
Xiaohong Deng ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Extraction Methods ◽  
Pcr Assay ◽  
Wide Range ◽  
The Mean ◽  
Salmonella Serotypes ◽  
Pathogen Dna ◽  
Dna Extraction Methods

ABSTRACTThe purpose of the study was to evaluate three real-time PCR platforms for rapid detection of Salmonella from cloves and to compare three different DNA extraction methods. Six trials were conducted with two clove cultivars, Ceylon and Madagascar, and three Salmonella serotypes, Montevideo, Typhimurium, and Weltevreden. Each trial consisted of 20 test portions. The preenrichment cultures were used to perform PCR for comparison of the effectiveness of U.S. Food and Drug Administration, Pacific Regional Laboratory Southwest (FDA-PRLSW), Applied Biosystems Inc. (ABI) MicroSEQ, and GeneDisc platforms for detection of Salmonella. Three DNA extraction methods were used: standard extraction method for each PCR platform, boil preparation, and LyseNow food pathogen DNA extraction cards. The results from real-time PCR correlated well with FDA Bacteriological Analytical Manual culture assay results, with a wide range of cycle threshold (CT) values among the three PCR platforms for intended positive samples. The mean CT values for MicroSEQ (16.36 ± 2.78) were significantly lower than for PRLSW (20.37 ± 3.45) and GeneDisc (23.88 ± 2.90) (P &lt; 0.0001). Pairwise comparisons between PCR platforms using different DNA extraction methods indicate that the CT values are inversely proportional to the relative DNA quantity (RDQ) yields by different platform-extraction combinations. The pairing of MicroSEQ and boil preparation generated the highest RDQ of 120 and the lowest average CT value of 14.48, whereas the pairing of GeneDisc and LyseNow generated the lowest RDQ of 0.18 and the highest average CT of 25.97. Boil preparation yielded higher RDQ than the other extraction methods for all three PCR platforms. Although the MicroSEQ platform generated the lowest CT values, its sensitivity was compromised by narrow separations between the positive and negative samples. The PRLSW platform generated the best segregation between positive and negative groups and is less likely to produce false results. In conclusion, FDA-PRLSW was the most efficient PCR assay for Salmonella detection, and boil preparation was the best method for DNA extraction.

Detection of Thermophilic Campylobacter spp. in Blood-Free Enriched Samples of Inoculated Foods by the Polymerase Chain Reaction

2000 ◽  
Vol 63 (3) ◽  
pp. 299-303 ◽  
Author(s):  
RICHARD L. THUNBERG ◽  
TONY T. TRAN ◽  
MARK O. WALDERHAUG
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Raw Milk ◽  
Extraction Techniques ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Enrichment Broth ◽  
Thermophilic Campylobacter ◽  
Polymerase Chain ◽  
Campylobacter Spp

The detection of thermophilic Campylobacter spp., as represented by Campylobacter jejuni, by the polymerase chain reaction (PCR) was investigated and compared with the selective agar isolation (SAI) method. Stationary-phase cultures of C. jejuni were inoculated into either blood-free enrichment broth (BFEB) or BFEB that contained 10% broccoli, crabmeat, mushroom, raw milk, and raw oyster rinses. Following a 48-h enrichment period, aliquots of food test portions were removed for simultaneous analysis by PCR and SAI. It was determined that the presence of charcoal and iron in the enrichment broth interfered with the PCR assay. Therefore, three DNA extraction techniques were developed and evaluated using a 16S rRNA primer pair in the PCR assay. The 50% end point (DL50) values (determined upon six initial C. jejuni spiking levels) were used to assess the frequency of isolation utilizing PCR versus SAI for the detection of this organism in the enrichment matrices. There were virtually no differences in detection of C. jejuni among enriched samples analyzed by PCR and SAI. Mean DL50 values (n = 3) for plain BFEB, broccoli, crabmeat, mushroom, raw milk, and raw oyster were, respectively, 0.02 (PCR) versus 0.01 (SAI), 0.01 versus 0.06, 0.07 versus 0.04, 0.03 versus 0.08, 0.01 versus 0.01, and 0.01 versus 0.01 CFU/5 g food. Significant variability in the detection limit of C. jejuni by PCR in the food enrichments was observed among DNA extraction techniques. Using 48-h enrichment cultures followed by PCR analysis could save 1 day of the time required for the presumptive identification of C. jejuni in suspected foods.

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