Hygienization by anaerobic digestion: comparison between evaluation by cultivation and quantitative real-time PCR

2005 ◽  
Vol 52 (1-2) ◽  
pp. 93-99 ◽  
Author(s):  
M. Lebuhn ◽  
M. Effenberger ◽  
G. Garcés ◽  
A. Gronauer ◽  
P.A. Wilderer
Keyword(s):  
Anaerobic Digestion ◽  
Real Time ◽  
Microbial Activity ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Full Scale ◽  
Quantitative Real Time Pcr ◽  
Highly Effective ◽  
The Difference

In order to assess hygienization by anaerobic digestion, a comparison between evaluation by cultivation and quantitative real-time PCR (qPCR) including optimized DNA extraction and quantification was carried out for samples from a full-scale fermenter cascade (F1, mesophilic; F2, thermophilic; F3, mesophilic). The system was highly effective in inactivating (pathogenic) viable microorganisms, except for spore-formers. Conventionally performed cultivation underestimated viable organisms particularly in F2 and F3 by a factor of at least 10 as shown by data from extended incubation times, probably due to the rise of sublethally injured (active but not cultivable) cells. Incubation should hence be extended adequately in incubation-based hygiene monitoring of stressed samples, in order to minimize contamination risks. Although results from qPCR and cultivation agreed for the equilibrated compartments, considerably higher qPCR values were obtained for the fermenters. The difference probably corresponded to DNA copies from decayed cells that had not yet been degraded by the residual microbial activity. An extrapolation from qPCR determination to the quantity of viable organisms is hence not justified for samples that had been exposed to lethal stress.

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.

Quantification of Cryptosporidium parvum in anaerobic digesters treating manure by (reverse-transcription) quantitative real-time PCR, infectivity and excystation tests

2006 ◽  
Vol 53 (8) ◽  
pp. 195-202 ◽  
Author(s):  
G. Garcés ◽  
M. Effenberger ◽  
M. Najdrowski ◽  
C. Wackwitz ◽  
A. Gronauer ◽  
...  
Keyword(s):  
Anaerobic Digestion ◽  
Real Time ◽  
Cryptosporidium Parvum ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Combined Treatment ◽  
Quantitative Real Time Pcr ◽  
Anaerobic Digesters ◽  
Hsp70 Mrna ◽  
Cryptosporidium Parvum Oocysts

The survival of Cryptosporidium parvum oocysts in anaerobic digesters treating manure was investigated for mesophilic, thermophilic, and a combined treatment (mesophilic–thermophilic–mesophilic) under different retention times of oocysts in the reactors. C. parvum DNA was extracted with an optimised protocol, and its amount determined by quantitative real-time PCR (qPCR). Results indicated noteworthy differences in DNA content after the different treatments. DNA was not degraded during the process. However, excystation and infectivity tests showed a reduction of viable oocyst numbers of ≥2 and ≥5 log units after the thermophilic treatment in two different experiments. Thus qPCR-targeting DNA can overestimate the number of oocysts that survive and remain viable after anaerobic digestion. However, targeting DNA is suitable to indicate the presence or absence of oocysts. Reverse transcription qPCR (RT-qPCR) targeting C. parvum hsp70 mRNA successfully indicated the presence of viable fraction of oocysts.

Using genes differentially expressed in bulls to classify steers divergently selected for high and low residual feed intake

2012 ◽  
Vol 52 (7) ◽  
pp. 608 ◽  
Author(s):  
Y. Chen ◽  
P. F. Arthur ◽  
R. M. Herd ◽  
K. Quinn ◽  
I. M. Barchia
Keyword(s):  
Real Time ◽  
Feed Intake ◽  
Real Time Pcr ◽  
Calcium Binding ◽  
Correlation Coefficients ◽  
Residual Feed Intake ◽  
Differentially Expressed ◽  
Quantitative Real Time Pcr ◽  
The Difference ◽  
Estimated Breeding Values

Feed efficiency is an economically important trait in livestock and residual feed intake (RFI) is a commonly used measure of the trait in beef cattle. Residual feed intake is the difference between the actual feed intake recorded over a test period and the expected feed intake of an animal based on its size and growth rate. It is a heritable trait, and efficient animals have lower RFI values. Several genes have been identified as being differentially expressed in the liver of Angus bulls that have been divergently selected for RFI. The objective of this study was to use genes that are differentially expressed in bulls to classify Angus steers from the same divergent RFI selection lines. Liver samples were collected at slaughter from 40 high RFI and 40 low RFI steers that were ~23 months old, and had just completed a 251-day feedlot feeding period. RNA samples from the livers were assayed by quantitative real-time PCR for 14 genes, which have been identified previously in bulls. Steers were not measured for RFI, hence the estimated breeding values (EBV) for RFI of their parents were used to calculate their mid-parent (average of the two parents) RFI-EBV. Correlation and discriminant analyses were conducted on the normalised quantitative real-time PCR data from the steers. Discriminant analysis was also conducted on the bull data for comparison. In the steers, 8 out of the 14 genes were significantly (P < 0.05) correlated with RFI-EBV. Two genes from the glutathione S-transferase mu family (GSTM1 and GSTM2) and the S100 calcium-binding protein A10 (S100A10) had the highest correlations with RFI-EBV, with correlation coefficients of 0.59, 0.44 and 0.36, respectively. Based on the 14 expressed genes, 84% of the steers and 98% of the bulls were correctly classified into their respective RFI selection lines. The results of this study indicate that a high proportion of the genes that were differentially expressed in the original study with bulls were also differentially expressed in this study with steers. The high accuracy in classification obtained in this study shows that the transcriptional approach to the study of the biological processes involved in variation in RFI has great potential for identification of candidate genes.

scholarly journals Automated and Manual Methods of DNA Extraction for Aspergillus fumigatus and Rhizopus oryzae Analyzed by Quantitative Real-Time PCR

2008 ◽  
Vol 46 (6) ◽  
pp. 1978-1984 ◽  
Author(s):  
A. Francesconi ◽  
M. Kasai ◽  
S. M. Harrington ◽  
M. G. Beveridge ◽  
R. Petraitiene ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Rhizopus Oryzae ◽  
Quantitative Real Time Pcr ◽  
Manual Methods

scholarly journals Improved DNA extraction and quantitative real-time PCR for genotyping Erysiphe necator and detecting the DMI fungicide resistance marker A495T, using single ascocarps

2020 ◽  
Vol 59 (1) ◽  
pp. 97-106
Author(s):  
Alexandra PINTYE ◽  
Márk Z. NÉMETH ◽  
Orsolya MOLNÁR ◽  
Áron N. HORVÁTH ◽  
Zsolt SPITZMÜLLER ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Fungicide Resistance ◽  
Infected Plant ◽  
Quantitative Real Time Pcr ◽  
Plant Materials ◽  
Erysiphe Necator ◽  
Genetic Groups ◽  
Field Samples

DNA extraction from minute fungal samples is challenging in all genetic studies. Identification of genetic groups and population biology mostly rely on the laborious production of single conidium isolates or on field samples, including infected plant materials. This paper reports a simple and cost-effective protocol for DNA extraction from individual chasmothecia of Erysiphe necator for subsequent applications. It is a less laborious alternative for genotyping purposes than production and analysis of single conidium isolates or analysis of infected plant material from the field. Using the protocols described here for 186 E. necator samples tested, genetic groups A and B were assigned. Based on CYP51 sequences, all the samples belonged to group B, while TUB2 sequences exhibited SNPs also diagnostic for group A. Additionally, a quantitative real-time PCR detection method of single nucleotide polymorphism in the CYP51 gene associated with DMI fungicide resistance was applied. The A495T marker, associated with DMI resistance, and here reported for the first time from Hungary, was detected by quantitative real-time PCR assays and direct sequencing of CYP51. The methods developed in this study can be applied as routine tests to monitor powdery mildew populations for fungicide resistance and other genetic characteristics.

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