scholarly journals Simultaneous Detection and Differentiation of Pathogenic and Nonpathogenic Leptospira spp. by Multiplex Real-Time PCR (TaqMan) Assay

2010 ◽  
Vol 59 (3) ◽  
pp. 167-173 ◽  
Author(s):  
ORHAN BEDIR ◽  
ABDULLAH KILIC ◽  
ERDINC ATABEK ◽  
AHMET MERT KUSKUCU ◽  
VEDAT TURHAN ◽  
...  
Keyword(s):  
Real Time ◽  
Ribosomal Rna ◽  
Real Time Pcr ◽  
Molecular Techniques ◽  
16S Ribosomal Rna ◽  
Taqman Assay ◽  
Pathogenic Leptospira ◽  
Ribosomal Rna Gene ◽  
Leptospira Spp ◽  
23S Ribosomal Rna

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10(2) cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.

scholarly journals Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis

PLoS ONE ◽  
2014 ◽  
Vol 9 (11) ◽  
pp. e112356 ◽  
Author(s):  
Jesse J. Waggoner ◽  
Ilana Balassiano ◽  
Janaki Abeynayake ◽  
Malaya K. Sahoo ◽  
Alisha Mohamed-Hadley ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Detection ◽  
Nucleic Acid Amplification ◽  
Pathogenic Leptospira ◽  
Leptospira Spp

scholarly journals The Effect of Environmental Stresses on lipL32 Gene Expression in Pathogenic Leptospira spp. through Real-Time PCR

2020 ◽  
Vol 69 (3) ◽  
pp. 301-310
Author(s):  
SONA ROSTAMPOUR YASOURI ◽  
MONIR DOUDI ◽  
MASOOD GHANE ◽  
NAFISEH SADAT NAGHAVI ◽  
ABOLHASAN REZAEI
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Plant Extract ◽  
Real Time Pcr ◽  
Northern Iran ◽  
Pathogenic Leptospira ◽  
Leptospira Spp ◽  
Different Temperatures ◽  
Green Technique

Leptospirosis is a worldwide infectious and zoonotic disease. The incidence of this disease is high in temperate regions, especially in northern Iran. The aim of this study was to investigate the effects of temperature, pH, and Phyllanthus amarus plant extract on the lipL32 gene expression in pathogenic Leptospira spp. Fifty water samples were collected. Culture and PCR technique were used to isolate and identify the bacterium and the presence of the lipL32 gene. The samples were exposed to different temperatures and pH levels for one day and the Ph. amarus plant extract at different concentrations for one and seven days. RNA was extracted, and cDNA synthesis was performed for all the samples. All cDNAs were evaluated by the real-time PCR (SYBR green) technique. Out of the 50 samples, ten samples (20%), using PCR were determined to contain the pathogenic Leptospira. Fold change of the expression of the lipL32 gene associated with stresses was as follows: temperature stress of 40°C, 35°C, and 25°C reduced the lipL32 gene expression in all three isolates, especially in the isolates type 1. The pH stress, i.e., pH values equal to 8 or 9 reduced the gene expression in three types of isolates, and pH = 6 stress increases the lipL32 gene expression in the isolates of type 1. Ph. amarus plant extract stress reduced the mentioned gene expression only in isolates of type 2. Temperature and pH stresses could lead to differences in the expression level and cause the lipL32 gene expression decrease in three pathogenic isolates. The MIC results showed anti-leptospiral effect of Ph. amarus plant extract.

scholarly journals Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions

2014 ◽  
Vol 110 (3) ◽  
pp. 165-172 ◽  
Author(s):  
Q Wu ◽  
KC Prager ◽  
T Goldstein ◽  
DP Alt ◽  
RL Galloway ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
California Sea Lions ◽  
Pathogenic Leptospira ◽  
Sea Lions ◽  
Leptospira Spp

scholarly journals Comparison of Real-Time PCR Assays for Detection of Pathogenic Leptospira spp. in Blood and Identification of Variations in Target Sequences

2011 ◽  
Vol 49 (6) ◽  
pp. 2154-2160 ◽  
Author(s):  
Pascale Bourhy ◽  
Sylvie Bremont ◽  
Farida Zinini ◽  
Claude Giry ◽  
Mathieu Picardeau
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogenic Leptospira ◽  
Leptospira Spp ◽  
Pcr Assays ◽  
Target Sequences

scholarly journals Molecular Approaches to Studying Microbial Communities: Targeting the 16S Ribosomal RNA Gene

2016 ◽  
Vol 38 (3) ◽  
pp. 223-232 ◽  
Author(s):  
Kazumasa FUKUDA ◽  
Midori OGAWA ◽  
Hatsumi TANIGUCHI ◽  
Mitsumasa SAITO
Keyword(s):  
Microbial Communities ◽  
Ribosomal Rna ◽  
16S Ribosomal Rna ◽  
Approaches To Studying ◽  
Ribosomal Rna Gene ◽  
Molecular Approaches ◽  
16S Ribosomal Rna Gene

scholarly journals Detection of Peanut Allergen Traces with a Real Time PCR Assay - The Challenge to Protect Food-Allergic Consumers

2017 ◽  
Vol 7 (1) ◽  
pp. 32 ◽  
Author(s):  
Dimitra Houhoula ◽  
Stamatios Koussissis ◽  
Vladimiros Lougovois ◽  
John Tsaknis ◽  
Dimitra Kassavita ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Food Products ◽  
Molecular Techniques ◽  
Food Allergens ◽  
Pcr Assay ◽  
Peanut Allergen ◽  
Pcr Method ◽  
Detection And Quantification

The aim of the present study was the implementation of molecular techniques in the detection and quantification of allergic substances of peanut in various kinds of food products, e.g., breakfast cereals, chocolates and biscuits that are frequently related to allergies. In some cases, the presence of peanuts can be due to contamination during production and are not declared on the label. A total of 152 samples were collected from supermarkets and were analysed by a Real Time PCR method. The results indicated that 125 samples (83,3%) were found positive in peanut traces but the most important finding is that from the 84 samples that had no allergen declaration for peanuts, 48 (57,1%) of them were found positive. In conclusion, Real Time PCR can be a very important tool for the rapid detection and quantification of food allergens.

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