PMID- 33035573
OWN - NLM
STAT- Publisher
LR  - 20240222
IS  - 1872-8359 (Electronic)
IS  - 0167-7012 (Linking)
VI  - 178
DP  - 2020 Oct 6
TI  - Development and application of a real-time PCR assay for the sensitive detection 
      of diarrheic toxin producer Prorocentrum lima.
PG  - 106081
LID - S0167-7012(20)30797-1 [pii]
LID - 10.1016/j.mimet.2020.106081 [doi]
AB  - Prorocentrum lima (P. lima) is a widely spread dinoflagellate in the 
      Mediterranean Sea and it has become increasingly involved in harmful algal 
      blooms. The purpose of this study is to develop a probe-based real-time 
      polymerase chain reaction (PCR) targeting the ITS1-5.8S-ITS2 region for the 
      detection and absolute quantification of P. lima based on linear and circular DNA 
      standards. The results have shown that the quantitative PCR (q-PCR), using 
      circular plasmid as a template, gave a threshold cycle number 1.79-5.6 greater 
      than equimolar linear standards. When microalgae, commonly found in aquatic 
      samples were tested, no cross-amplification was observed. The q-PCR brought about 
      a good intra and inter-run reproducibility and a detection limit of 5 copies of 
      linear plasmid per reaction. A quantitative relationship between the cell numbers 
      and their corresponding plasmid copy numbers was attained. Afterwards, the 
      effectiveness of the developed protocol was tested with 130 aquatic samples taken 
      from 19 Tunisian sampling sites. The developed q-PCR had a detection sensitivity 
      of up to 1 cell. All the positive samples were taken from three sampling sites of 
      Medenine Governorate with cell abundances that ranged from 22 to 156,000 cells 
      L(-1) of seawater. The q-PCR assay revealed a high sensitivity in monitoring the 
      aquatic samples in which the low concentrations of P. lima were not accurately 
      detected by light microscopy. Indeed, this approach is at the same time rapid, 
      specific and sensitive than the traditional microscopy techniques and it 
      represents a great potential for the monitoring of P. lima blooms.
CI  - Copyright (c) 2020 Elsevier B.V. All rights reserved.
FAU - Barkallah, Mohamed
AU  - Barkallah M
AD  - Laboratoire de Genie Enzymatique et Microbiologie, Equipe de Biotechnologie des 
      Algues, Ecole Nationale d'Ingenieurs de Sfax, University of Sfax, 3038 Sfax, 
      Tunisia.
FAU - Elleuch, Jihen
AU  - Elleuch J
AD  - Laboratoire de Genie Enzymatique et Microbiologie, Equipe de Biotechnologie des 
      Algues, Ecole Nationale d'Ingenieurs de Sfax, University of Sfax, 3038 Sfax, 
      Tunisia.
FAU - Smith, Kirsty F
AU  - Smith KF
AD  - Cawthron Institute, 98 Halifax Street East, Private Bag 2, Nelson 7042, New 
      Zealand.
FAU - Chaari, Siwar
AU  - Chaari S
AD  - Laboratoire de Genie Enzymatique et Microbiologie, Equipe de Biotechnologie des 
      Algues, Ecole Nationale d'Ingenieurs de Sfax, University of Sfax, 3038 Sfax, 
      Tunisia.
FAU - Ben Neila, Idriss
AU  - Ben Neila I
AD  - Veterinary Research Center of Sfax, Sfax, Tunisia.
FAU - Fendri, Imen
AU  - Fendri I
AD  - Laboratory of Plant Biotechnology Applied to the improvement of Cultures, Faculty 
      of Sciences of Sfax, B.P. 1171, 3000, University of Sfax, 3029 Sfax, Tunisia.
FAU - Michaud, Philippe
AU  - Michaud P
AD  - Universite Clermont Auvergne, CNRS, SIGMA Clermont, Institut Pascal, F-63000 
      Clermont-Ferrand, France.
FAU - Abdelkafi, Slim
AU  - Abdelkafi S
AD  - Laboratoire de Genie Enzymatique et Microbiologie, Equipe de Biotechnologie des 
      Algues, Ecole Nationale d'Ingenieurs de Sfax, University of Sfax, 3038 Sfax, 
      Tunisia. Electronic address: slim.abdelkafi@enis.tn.
LA  - eng
PT  - Journal Article
DEP - 20201006
PL  - Netherlands
TA  - J Microbiol Methods
JT  - Journal of microbiological methods
JID - 8306883
SB  - IM
OTO - NOTNLM
OT  - Absolute quantification
OT  - Bivalve molluscs
OT  - Hydrolysis probe
OT  - Toxic microalgae
COIS- Declaration of Competing Interest The authors declare that they have no conflict 
      of interest.
EDAT- 2020/10/10 06:00
MHDA- 2020/10/10 06:00
CRDT- 2020/10/09 20:09
PHST- 2020/08/28 00:00 [received]
PHST- 2020/10/01 00:00 [revised]
PHST- 2020/10/01 00:00 [accepted]
PHST- 2020/10/10 06:00 [pubmed]
PHST- 2020/10/10 06:00 [medline]
PHST- 2020/10/09 20:09 [entrez]
AID - S0167-7012(20)30797-1 [pii]
AID - 10.1016/j.mimet.2020.106081 [doi]
PST - aheadofprint
SO  - J Microbiol Methods. 2020 Oct 6;178:106081. doi: 10.1016/j.mimet.2020.106081.