A duplex PCR method for identification of cultures of Fusarium graminearum from infected wheat grain without DNA extraction

2018 ◽  
Vol 40 (3) ◽  
pp. 417-422 ◽  
Author(s):  
Krista Zuzak ◽  
Kher Zahr ◽  
Yalong Yang ◽  
Alian Sarkes ◽  
David Feindel ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Fusarium Graminearum ◽  
Duplex Pcr ◽  
Wheat Grain ◽  
Pcr Method

An Efficient Duplex PCR Method for Sex Identification of the European Grapevine Moth Lobesia botrana (Lepidoptera: Tortricidae) at Any Developmental Stage

2020 ◽  
Vol 113 (5) ◽  
pp. 2505-2510
Author(s):  
Carlos Aguirre ◽  
Natalia Olivares ◽  
Patricio Hinrichsen
Keyword(s):  
Developmental Stage ◽  
Lobesia Botrana ◽  
Sex Identification ◽  
Morphological Identification ◽  
Sequence Information ◽  
Duplex Pcr ◽  
W Chromosome ◽  
Starting Point ◽  
Pcr Method ◽  
Specific Sequences

Abstract Many genetic studies in insects require sex identification of individuals in all developmental stages. The most common sex chromosome system in lepidopterans is WZ/ZZ; the W chromosome is present only in females. Based on two W chromosome-specific short sequences (CpW2 and CpW5) described in Cydia pomonella (L.) (Lepidoptera: Tortricidae), we identified homologous female-specific sequences in Lobesia botrana Den. & Schiff, a polyphagous and very harmful species present in Chile since 2008. From this starting point, we extended the sequence information using the inverse PCR method, identifying the first W-specific sequences described up to now for the moth. Finally, we developed a duplex PCR method for rapid and sensitive determination of sex in L. botrana from larva to adult. The method showed a detection limit of 1 pg of genomic DNA; a blind panel of samples exhibited exact correspondence with the morphological identification. These results will be very useful for studies requiring sex-specific analyses at any developmental stage, contributing also to the understanding of gene expression in the insect, as well as to the eventual development of control protocols against the moth, such as the development of genetic sexing strains for the implementation of the sterile insect technique.

Quantitative Detection of Campylobacter jejuni on Fresh Chicken Carcasses by Real-Time PCR

2007 ◽  
Vol 70 (6) ◽  
pp. 1373-1378 ◽  
Author(s):  
ANNA-CLARA RÖNNER ◽  
HANS LINDMARK
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Dna Extraction ◽  
Campylobacter Jejuni ◽  
Real Time Pcr ◽  
Close Correlation ◽  
Quantitative Detection ◽  
Quantitative Real Time Pcr ◽  
Direct Plating ◽  
Pcr Method

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 μl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

Detection of Rendered Meat and Bone Meals by PCR Is Dependent on Animal Species of Origin and DNA Extraction Method

2010 ◽  
Vol 73 (6) ◽  
pp. 1090-1096 ◽  
Author(s):  
MICHAEL J. MYERS ◽  
DOROTHY E. FARRELL ◽  
CHRISTINE M. DEAVER ◽  
JACQULINE MASON ◽  
HEIDI L. SWAIM ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Animal Species ◽  
Animal Feed ◽  
Functional Assay ◽  
Threshold Values ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Pcr Method

The capability of eight commercially available DNA extraction kits to extract bovine DNA originating in meat and bone meal from fortified feed was evaluated. Four different batches of bovine meat and bone meal (BMBM) were used for DNA extraction with the eight commercial DNA extraction kits. Within each kit, there were minimal differences in the batch-to-batch amounts of extracted DNA. There were differences between the kits in the amounts of DNA that could be extracted from the same amount of starting BMBM. These differences did not translate into differences in the amount of amplifiable DNA from BMBM-fortified dairy feed. Using a validated real-time PCR method, the kit yielding the highest amount extractable DNA was completely unable to yield a positive PCR result; one other kit was also unable to produce a positive PCR result from DNA extracted from BMBM-fortified feed. There was a complete lack of a correlation between the amount of bovine DNA isolated from BMBM by a given extraction kit compared with the relative amounts of DNA isolated from fortified animal feed as evidenced by the cycle threshold values generated using the real-time PCR method. These results demonstrate that extraction of DNA from processed animal protein is different for pure ingredients and fortified animal feeds. These results indicate that a method specifically developed using just animal-derived meat and bone meal may not yield a functional assay when used to detect animal tissues in complete animal feed.

scholarly journals COMPARISON OF METHODS FOR DETECTION OF MICROSPORIDIA SPECIES OF THE GENUS NOSEMA IN HONEY BEES (APIS MELLIFERA)

2013 ◽  
Vol 6 (1) ◽  
pp. 19-27
Author(s):  
Uroš Glavinić ◽  
Aleksandar Stanković ◽  
Jevrosima Stevanović ◽  
Predrag Simeunović ◽  
Nevenka Aleksić ◽  
...  
Keyword(s):  
Honey Bees ◽  
Pcr Analysis ◽  
Duplex Pcr ◽  
Infection Level ◽  
Nosema Species ◽  
Pcr Method ◽  
Biological Methods ◽  
Pcr Techniques ◽  
Microsporidia Species

Two microsporidia species of the Nosema genus cause nosemosis in the adult honeybee: N. apis and N. ceranae. For diagnostic purposes and the determination of infection level various microscopic and molecular biological methods are used. Th e aim of this research was to compare the reliability of the traditional microscopic assessment and two PCR techniques: simplex- and duplex-PCR. Honey bee samples were taken from 150 colonies. Microscopic examination, performed according to the recommendations of the OIE, revealed Nosema spores in 68.67% samples analysed, whilst with the simplex-PCR method all samples (100.0%) proved positive. On the other hand, duplex-PCR method used for the identifi cation of Nosema species resulted in 84.0% positive samples, all of which were N. ceranae. Our recommendation of the simplex-PCR method for the monitoring of honey bees in fi eld conditions is based on its higher reliability than the microscopic assessment in the detection of low-level infections, as well as its potential for the detection of vegetative Nosema sp. stages; thus the early detection and timely prevention of Nosema infection would be possible. Nosema species identifi cation is simplest and most cost-eff ective if performed with the duplex-PCR analysis. However, the simplex-PCR is more reliable, thus, it is suggested that samples that were negative when assessed with microscopy and duplex-PCR analysis undergo simplex-PCR.

scholarly journals Evaluation of the performance of an in-house duplex PCR assay targeting the IS6110 and rpoB genes for tuberculosis diagnosis in Cameroon

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Henry Dilonga Meriki ◽  
Ndze Henry Wung ◽  
Kukwah Anthony Tufon ◽  
Nyeke James Tony ◽  
Irene Ane-Anyangwe ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Low Income ◽  
Sodium Hydroxide Solution ◽  
Low Income Countries ◽  
Health Concern ◽  
Smear Microscopy ◽  
Duplex Pcr ◽  
Pcr Inhibition ◽  
Likelihood Ratios ◽  
Predictive Values

Abstract Background Tuberculosis (TB) remains a major public health concern in many low-income countries accounting for approximately two-thirds of deaths in people living with human immunodeficiency virus (HIV) infection. With prompt, accurate and appropriate treatment, almost all TB disease can be cured. The present study was to evaluate the diagnostic performance of an in-house duplex PCR (D-PCR) using IS1610 and rpoB specific primers in sputum samples from TB suspected patients. Methods A hospital-based cross-sectional study was conducted at the Limbe and Buea Regional Hospitals of the South West Region of Cameroon from June 2016 to April 2017. Sputum samples, decontaminated with hypertonic saline/sodium hydroxide solution were centrifuged and pellets processed for smear microscopy, culture and DNA extraction. Suspected inhibition was resolved by serial dilution of genomic DNA. Results were compared to culture as gold standard as well as a Composite Reference Standard (CRS). Results A total of 129 participants aged between 5 to 82 years were enrolled in to the study. The median age of the participants was 37 years (interquartile range, IQR: 27–50 years), with 54.3% being male. Forty-seven samples (36.4%) were positive by direct sputum microscopy, 49 (38%) by microscopy after concentration, 51 (39.5%) by culture and 62 (40.1%) by D-PCR. PCR inhibition was resolved in 85.7% (18/21) of the samples that had inhibition. The overall sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios and area under the curve AUC) of the D-PCR was 93.5, 94, 94, 94%, 15.6, 0.005 and 89.0% respectively using CRS as reference. The sensitivities of D-PCR observed among different sample categories were 95.7, 87.5 and 87.5% for smear-and culture-positives, smear-negative/culture-positive, and clinically diagnosed cases respectively. Conclusion IS1610 and rpoB duplex PCR using relatively cheap decontamination and DNA extraction methods in addition to simple serial dilutions to resolve PCR inhibition shows high sensitivity in the diagnosis of paucibacillary tuberculosis.

Identification and authentication of commercial mi-iuy croaker (Miichthys miiuy) products by two PCR-based methods

2020 ◽  
Author(s):  
tae sun kang
Keyword(s):  
Economic Value ◽  
Cost Effective ◽  
Cross Reactivity ◽  
Duplex Pcr ◽  
Consensus Sequences ◽  
Pcr Product ◽  
Otolithes Ruber ◽  
Pcr Method ◽  
First Time ◽  
Miichthys Miiuy

Mi-iuy croaker (Miichthys miiuy) is one of the most important ingredients of Korean cuisine and thus has the highest economic value. However, the similar morphological traits among croaker fish belonging to family Sciaenidae are often exploited for seafood fraud. In this study, M. miiuy-specific primer set was designed and further improved by the development of a rapid and cost-effective duplex PCR method. The specificity of M. miiuy-specific duplex PCR was tested using 22 seafood species, and no cross-reactivity was observed. The sensitivity of the PCR assay was found to be 0.1 ng/µL. For the first time, labeling compliance of 43 commercial m-iuy croaker products was verified using both full DNA barcoding and M. miiuy-specific duplex PCR methods. For species identification, BOLDSYSTEMS and GenBank database were screened with the consensus sequences of each PCR product as a query. This identification result was further confirmed using the M. miiuy-specific duplex PCR method. The findings of this study revealed that principal species substituted were law croaker (Pseudotolithus senegallus, n=4), bigeye croaker (Micropogonias megalops, n=3), whitemouth croaker (Micropogonias furnieri, n=1), and tigertoothed croaker (Otolithes ruber, n=1). A significant percentage (21%) of mislabeling was present in commercial mi-iuy products sold on the South Korea market.

scholarly journals Comparison of real-time PCR and the Kato-Katz method for the diagnosis of soil-transmitted helminthiasis and assessment of cure in a randomized controlled trial

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Beatrice Barda ◽  
Christian Schindler ◽  
Rahel Wampfler ◽  
Shaali Ame ◽  
Said M. Ali ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Controlled Trial ◽  
Bead Beating ◽  
Novel Treatments ◽  
Two Samples ◽  
Stool Samples ◽  
Pcr Method

Abstract Background Diagnosis of soil-transmitted helminths (STHs) in developing countries is commonly based on microscopic detection of eggs in stool samples, using the Kato-Katz (KK) method, which has a poor sensitivity for detecting light intensity infections. We compared the performance of the KK method and real-time PCR in the framework of a randomized trial, which evaluated four novel treatments against Trichuris trichiura and concomitant STH infections. Results Two stool samples obtained from 320 participants were examined at baseline and follow-up with quadruplicate KK and PCR analyses of one of the two samples using “bead-beating” for DNA extraction. At follow-up, 80 samples were negative according to both PCR and KK and 173 were positive with both methods for any of the STHs. Relative to PCR, the calculated sensitivity of KK at follow-up was 83.6%, 43.0% and 53.8% for T. trichiura, for hookworm and for Ascaris lumbricoides, respectively. The sensitivity of PCR compared with KK at this time point was 89.1% for T. trichiura, 72.7% for hookworm and 87.5% for A. lumbricoides. Cure rates (CRs) for T. trichiura and A. lumbricoides were slightly lower with the PCR method. For hookworm CRs with KK were mostly significantly lower, namely 36.7%, 91.1%, 72.2% and 77.8% for moxidectin, moxidectin in combination with tribendimidine, moxidectin in combination with albendazole and albendazole in combination with oxantel pamoate, respectively, whereas with PCR the CRs were 8.3%, 82.6%, 37.1% and 57.1%, respectively. Conclusions In conclusion, a single real-time PCR is as sensitive as quadruplicate KK for T. trichiura and A. lumbricoides detection but more sensitive for hookworm, which has an influence on the estimated treatment efficacy. PCR method with DNA extraction using the “bead-beating protocol” should be further promoted in endemic areas and laboratories that can afford the needed equipment. The study is registered at ISRCTN (no. 20398469).

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