enough to detect the pathogen in asymptomatic fish Osorio et al 1999 In compar

Enough to detect the pathogen in asymptomatic fish

This preview shows page 234 - 236 out of 594 pages.

enough to detect the pathogen in asymptomatic fish (Osorio et al, 1999). In compar- ison, the PCR-RFLP approach detected < 180 fg of purified DNA, and was useful for use with sea bream and sea bass (Zappulli et al, 2005). In one case, it was necessary to discriminate subspecies piscicida from damselae, which was achieved using TCBS on which the former did not grow (Rajan et al., 2003). However, this had been previously resolved by Osorio et al. (2000), who used multiplex-PCR to detect and differentiate subsp. damselae dind piscicida. The former produced two ampHfication products, i.e. of 267 (fragment of 16S rRNA) and 448 bp (fragment o^ureC gene) whereas the latter revealed only the 267 bp product. This suggests—and was confirmed by dot blot hybridisation—that subsp. piscicida lacks the ureC gene. A PCR has been developed for the detection of Ps. anguilliseptica, with a detection limit of 170-200 cells/PCR tube within 8h (Blanco et al., 2002). Molecular methods have been invoked to improve the identification of V. anguil- larum. Using partial 16S rRNA sequences, a specific 16S rRNA oligonucleotide probe detected a minimum of 5 x 10^ cells/ml in culture or tissue extracts (Rehnstam et al., 1989). Detection of 1-10 bacterial cells in culture or 10-100 cells (equivalent to 2 X 10^ to 2 X lO"^ cells/g of tissue) in turbot tissue per PCR reaction was detailed by Powell and Loutit (1994a) who used a 310 bp DNA fragment as a probe for V. anguillarum. This system detected 100 (but not 10) ng of purified genomic DNA of most serogroups (but not serogroup 07) of V. anguillarum, but not did react with other vibrios. Using the species-specific Gonzalez et al. (2003). probe in combination with membrane filtration, V. anguillarum could be detected in water (Powell and Loutit, 1994b). In a parallel development, an oligonucleotide (VaV3) detected 150ng by DNA:DNA slot blot hybridisation. The system did not cross- react with other species, and was capable of detecting 8 out of 10 serogroups of V. anguillarum (Martinez-Picardo et al., 1994). A high level of specificity and sensi- tivity (detection limit = 4.0 x 10^ cells/ml) involved the use of the toxR gene for the
Image of page 234
Diagnosis 213 detection of V. harveyi; the PCR of which took <5h to enact and selectively recognised 20 authentic representatives of the taxon (including cells in diseased fish tissues), but not representatives of other vibrios (Pang et al, 2006). PCR technology offers promise for the detection of Piscirickettsia salmonis (e.g. Heath et al, 2000; Mauel and Fryer, 2001; Venegas et al, 2004), and a system has already been designed which is capable of detecting 1 tissue culture infectious dose (Mauel et ai, 1996) and 1-10 genome equivalents (competitive PCR; Heath et ai, 2000). Subsequently, a PCR was described that was effective with a few milHHtres of serum (Marshall et al, 1998). The benefit of this system is that the test could be carried out on live fish, such as valuable broodstock. A TaqMdin PCR was specific and sensitive (0.5 TCIDso/ml) (Corbeil et al, 2003).
Image of page 235
Image of page 236

You've reached the end of your free preview.

Want to read all 594 pages?

  • Spring '20
  • Bacteria, representative, gram-negative bacteria

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern

Stuck? We have tutors online 24/7 who can help you get unstuck.
A+ icon
Ask Expert Tutors You can ask You can ask You can ask (will expire )
Answers in as fast as 15 minutes
A+ icon
Ask Expert Tutors