673
primers, which generated PCR of 207 bp and 953 bp, respectively, and the resultant assay detected bacterial genomic DNA in brain and posterior kidney of
infected Nile tilapia (Lukkana et al. 2014 ).
• A highly specifi c microarray was constructed based on coupling a 16S rDNA
PCR with DNA hybridization that permitted the simultaneous detection and differentiation of Aer. hydrophila, Edw. tarda, Fla. columnare, Lactococcus garvieae, Ph. damselae, Ps. anguilliseptica, Str. iniae and V. anguillarum (Chang
et al. 2012 ). Targets on the probes were reacted with streptavidin-conjugated
alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3′indolylphosphate, p -toluidine salt resulting in the formation of easily visible blue
spots. The sensitivity was 1 pg for genomic DNA and 10
3 CFU/ml for pure cultures (Chang et al. 2012 ).
• The simultaneous recognition of fi ve bacterial fi sh pathogens, i.e. Ph. damselae,
Ps. baetica, T. maritimum, T. soleae and V. harveyi, was achieved by reverse line
blot hybridization. Species-specifi c probes targeted the 16S-23S intergenic
spacer region (ISR) or the 23S rRNA gene leading to correct identifi cations with
sensitivity of the assay ranging from 1- 100 pg of genomic DNA of the pure
culture. (Lopez et al. 2012 ).
• A multiple PCR RNA polymerase using the ß subunit ( rpoB ) gene differentiated
Ph. damselae, V. harveyi and V. ichthyoenteri in olive fl ounder with detection
limits in kidney of 2.5 × 10
6 CFU/g, 2.5 × 10
4 CFU/g and 2.5 × 10
5 CFU/g, respectively (Kim et al. 2014a ).
• A real-time fl uorogenic loop-mediated isothermal amplifi cation assay integrated
on a microfl uidic disc chip was capable of simultaneously detecting 10 pathogens, i.e., Noc. seriolae, Ps. putida, Str. iniae, V. alginolyticus, V. anguillarum, V.
fl uvialis, V. harveyi, V. parahaemolyticus, V. rotiferianus and V. vulnifi cus . The
assay was well automated with only one pipetting operation per chip for dispensing the sample. The assay was reproducible and specifi c, and the limits of detection were from 0.40 to 6.42 pg/1.414 μl with a reaction time of <30 min (Zhou
et al. 2014 ).
The ability to discriminate reliably between multiple pathogens is a major
improvement on previous approaches. The extreme sensitivity should be a bonus,
but could give positive readings in the absence of clinical disease leading to control
measures, which might not always be needed.
Phenotypic Tests
For many pathogens emphasis has been placed on conventional phenotypic tests for
diagnosis, although in many laboratories there has been a rapid move towards
molecular methods. Nevertheless, phenotypic methods are still used extensively.
Caution is advocated should consideration be given to using commercially available
diagnostic kits. The API 20E and API-ZYM, API 20NE API 50CH, API 50 L,
Phenotypic Tests
primers, which generated PCR of 207 bp and 953 bp, respectively, and the resultant assay detected bacterial genomic DNA in brain and posterior kidney of
infected Nile tilapia (Lukkana et al. 2014 ).
• A highly specifi c microarray was constructed based on coupling a 16S rDNA
PCR with DNA hybridization that permitted the simultaneous detection and differentiation of Aer. hydrophila, Edw. tarda, Fla. columnare, Lactococcus garvieae, Ph. damselae, Ps. anguilliseptica, Str. iniae and V. anguillarum (Chang
et al. 2012 ). Targets on the probes were reacted with streptavidin-conjugated
alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3′indolylphosphate, p -toluidine salt resulting in the formation of easily visible blue
spots. The sensitivity was 1 pg for genomic DNA and 10
3 CFU/ml for pure cultures (Chang et al. 2012 ).
• The simultaneous recognition of fi ve bacterial fi sh pathogens, i.e. Ph. damselae,
Ps. baetica, T. maritimum, T. soleae and V. harveyi, was achieved by reverse line
blot hybridization. Species-specifi c probes targeted the 16S-23S intergenic
spacer region (ISR) or the 23S rRNA gene leading to correct identifi cations with
sensitivity of the assay ranging from 1- 100 pg of genomic DNA of the pure
culture. (Lopez et al. 2012 ).
• A multiple PCR RNA polymerase using the ß subunit ( rpoB ) gene differentiated
Ph. damselae, V. harveyi and V. ichthyoenteri in olive fl ounder with detection
limits in kidney of 2.5 × 10
6 CFU/g, 2.5 × 10
4 CFU/g and 2.5 × 10
5 CFU/g, respectively (Kim et al. 2014a ).
• A real-time fl uorogenic loop-mediated isothermal amplifi cation assay integrated
on a microfl uidic disc chip was capable of simultaneously detecting 10 pathogens, i.e., Noc. seriolae, Ps. putida, Str. iniae, V. alginolyticus, V. anguillarum, V.
fl uvialis, V. harveyi, V. parahaemolyticus, V. rotiferianus and V. vulnifi cus . The
assay was well automated with only one pipetting operation per chip for dispensing the sample. The assay was reproducible and specifi c, and the limits of detection were from 0.40 to 6.42 pg/1.414 μl with a reaction time of <30 min (Zhou
et al. 2014 ).
The ability to discriminate reliably between multiple pathogens is a major
improvement on previous approaches. The extreme sensitivity should be a bonus,
but could give positive readings in the absence of clinical disease leading to control
measures, which might not always be needed.
Phenotypic Tests
For many pathogens emphasis has been placed on conventional phenotypic tests for
diagnosis, although in many laboratories there has been a rapid move towards
molecular methods. Nevertheless, phenotypic methods are still used extensively.
Caution is advocated should consideration be given to using commercially available
diagnostic kits. The API 20E and API-ZYM, API 20NE API 50CH, API 50 L,
Phenotypic Tests