scholarly journals Development of a Taqman real-time PCR assay for rapid detection and quantification ofVibrio tapetisin extrapallial fluids of clams

PeerJ ◽  
2015 ◽  
Vol 3 ◽  
pp. e1484 ◽  
Author(s):  
Adeline Bidault ◽  
Gaëlle G. Richard ◽  
Cédric Le Bris ◽  
Christine Paillard

The Gram-negative bacteriumVibrio tapetisis known as the causative agent of Brown Ring Disease (BRD) in the Manila clamVenerupis(=Ruditapes)philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification ofV. tapetisstrains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600TV. tapetisstrain. Quantification curves ofV. tapetisstrain seeded in filtered seawater (FSW) or extrapallial fluids (EF) samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detectV. tapetisstrains down to 1.125 101bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitorV. tapetisload both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections byV. tapetisand for designing appropriate control measures for aquaculture purposes.

2016 ◽  
Vol 55 (2) ◽  
pp. 180-184 ◽  
Author(s):  
Solène Le Gal ◽  
Florence Robert-Gangneux ◽  
Yann Pépino ◽  
Sorya Belaz ◽  
Céline Damiani ◽  
...  

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.


2021 ◽  
pp. 101729
Author(s):  
Guoying Dong ◽  
Qian Wang ◽  
Jiangting Niu ◽  
Yiqin Cai ◽  
Yanbing Guo ◽  
...  
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