Immuno-capture PCR method for detecting Acidovorax avenae subsp. citrulli from watermelon

2007 ◽  
Vol 4 (2) ◽  
pp. 173-179 ◽  
Author(s):  
Wang Xiao ◽  
Zhang Le ◽  
Xu Fu-Shou ◽  
Zhao Li-Han ◽  
Xie Guan-Lin
Keyword(s):  
Polymerase Chain Reaction ◽  
Low Cost ◽  
Chain Reaction ◽  
Direct Pcr ◽  
Causal Organism ◽  
Bacterial Fruit Blotch ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Honeydew Melon ◽  
Melon Seeds

AbstractAn immuno-capture polymerase chain reaction (IC-PCR) method for detection of Acidovorax avenae subsp. citrulli (AAC), the causal organism of bacterial fruit blotch (BFB) of watermelon, was developed by combining the immunosorbent enrichment (ISE) method with classical PCR and comparing with the direct PCR and growth check methods. The results showed that all A. avenae subsp. citrulli strains tested have produced 360 bp specific fragments using IC-PCR and direct PCR methods, while other strains from 10 different genera showed negative PCR results. The minimum detection concentration was about 50–100 cfu/ml and 104 cfu/ml, respectively. The IC-PCR sensitivity was 100 times higher than that of direct PCR. The examination of seven batches of different melon seeds from the markets by IC-PCR showed that one cantaloupe, two honeydew melon and two watermelon seed varieties carried the pathogen, indicating that the IC-PCR is an accurate, sensitive, rapid and low-cost technique.

Occurrence of Brachyspira hyodysenteriae in multiplier pig herds in Switzerland

2016 ◽  
Vol 44 (01) ◽  
pp. 13-18 ◽  
Author(s):  
S. Löbert ◽  
W. Zimmermann ◽  
S. Bürki ◽  
J. Frey ◽  
H. Nathues ◽  
...  
Keyword(s):  
Risk Factors ◽  
Polymerase Chain Reaction ◽  
Pilot Study ◽  
External Validation ◽  
Kappa Index ◽  
Chain Reaction ◽  
Direct Pcr ◽  
Brachyspira Hyodysenteriae ◽  
Pcr Method ◽  
Polymerase Chain

SummaryObjective: This research was aimed to determine the occurrence of Brachyspira (B.) hyodysenteriae in Swiss multiplier pig herds. Materials and methods: In a pilot study a direct real-time polymerase chain reaction (PCR) method for B. hyodysenteriae was compared to culture followed by PCR on 106 samples from three herds. Subsequently 40 multiplier herds were epidemiologically characterized and analysed for the presence of B. hyodysenteriae using direct PCR on 1412 rectal swabs. For external validation 20 swabs obtained from two positive conventional herds were analysed. Results: The comparison of direct PCR with culture followed by PCR resulted in a moderate agreement (kappa index: 0.58). In the two conventional herds, 35% of the samples (7/20) tested positive. Samples from 39 multipliers tested negative. In one multiplier herd, 25% (9/36) of the samples tested PCR positive. Risk factors in the multiplier herd may have been rodents or birds, but not pig purchase. Conclusion and clinical relevance: B. hyodysenteriae have been detected in a Swiss multiplier herd, which underlines the threat of potential spread by replacement pigs. Consequently, a Brachyspira monitoring programme was established for Swiss multiplier herds.

scholarly journals Design and development of polymerase chain reaction thermal cycler using proportional-integral temperature controller

2018 ◽  
Vol 14 (2) ◽  
pp. 213-218
Author(s):  
Chong Kim Soon ◽  
Nawoor Anusha Devi ◽  
Kok Beng Gan ◽  
Sue-Mian Then
Keyword(s):  
Polymerase Chain Reaction ◽  
Low Cost ◽  
Maximum Temperature ◽  
Gradient System ◽  
Chain Reaction ◽  
Temperature Controller ◽  
Proportional Integral ◽  
Integral Temperature ◽  
Polymerase Chain ◽  
Thermal Cycler

A thermal cycler is used to amplify segments of DNA using the polymerase chain reaction (PCR). It is an instrument that requires precise temperature control and rapid temperature changes for certain experimental protocols. However, the commercial thermal cyclers are still bulky, expensive and limited for laboratory use only.  As such it is difficult for on-site molecular screening and diagnostics. In this work, a portable and low cost thermal cycler was designed and developed. The thermal cycler block was designed to fit six microcentrifuge tubes. A Proportional-Integral temperature controller was used to control the thermal cycler block temperature. The results showed that the maximum temperature ramp rate of the developed thermal cycler was 5.5 °C/s. The proportional gain (Kp) and integral gain (Ki) of the PI controller were 15 A/V and 1.8 A/Vs respectively. Finally, the developed thermal cycler successfully amplified six DNA samples at the expected molecular weight of 150 base pair. It has been validated using the Eppendorf Mastercycler nexus gradient system and gel electrophoresis analysis

scholarly journals Diversity ofLeptospiraspp. in Rats and Environment from Urban Areas of Sarawak, Malaysia

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Chai Fung Pui ◽  
Lesley Maurice Bilung ◽  
Kasing Apun ◽  
Lela Su’ut
Keyword(s):  
Polymerase Chain Reaction ◽  
Water Samples ◽  
Urban Areas ◽  
Environmental Samples ◽  
Soil Samples ◽  
Chain Reaction ◽  
Prevalence Studies ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Soil And Water

Various prevalence studies onLeptospirain animals and humans, as well as environmental samples, had been conducted worldwide, including Malaysia. However, limited studies have been documented on the presence of pathogenic, intermediate, and saprophyticLeptospirain selected animals and environments. This study was therefore conducted to detectLeptospiraspp. in rats, soil, and water from urban areas of Sarawak using the polymerase chain reaction (PCR) method. A total of 107 rats, 292 soil samples, and 324 water samples were collected from April 2014 to February 2015. PathogenicLeptospirawas present in 5.6% (6/107) of rats, 11.6% (34/292) of soil samples, and 1.9% (6/324) of water samples. IntermediateLeptospirawas present in 2.7% (8/292) of soil samples and 1.9% (6/324) of water samples. SaprophyticLeptospirawas present in 10.3% (11/107) of rats, 1.4% (4/292) of soil samples, and 0.3% (1/324) of water samples. From this study, 76Leptospiraspp. were isolated. Based on DNA sequencing, the dominantLeptospiraspp. circulating in urban areas of Sarawak are pathogenicLeptospira noguchii, intermediateLeptospira wolffiiserovar Khorat, and saprophyticLeptospira meyeri, respectively. Overall, this study provided important surveillance data on the prevalence ofLeptospiraspp. from rats and the environment, with dominant local serovars in urban areas of Sarawak.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

Sign in / Sign up

Export Citation Format

Share Document