Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT‐PCR plus nested PCR

Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT‐PCR... A polymerase chain reaction (PCR)‐based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one‐step reverse‐transcription (RT)‐PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100‐fold when carried out in a separate tube (two‐step assay) and 10‐fold when performed in the same tube (one‐step assay). The sensitivity of the two‐step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT‐PCR alone but only by the two‐step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV‐positive broodfish by blood sampling and testing of their larval progeny. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Fish Diseases Wiley

Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT‐PCR plus nested PCR

Loading next page...
 
/lp/wiley/development-of-a-sensitive-diagnostic-assay-for-fish-nervous-necrosis-TxNZQ4y1T7
Publisher
Wiley
Copyright
Copyright © 2000 Wiley Subscription Services, Inc., A Wiley Company
ISSN
0140-7775
eISSN
1365-2761
D.O.I.
10.1046/j.1365-2761.2000.00255.x
Publisher site
See Article on Publisher Site

Abstract

A polymerase chain reaction (PCR)‐based assay to detect nervous necrosis virus (NNV) in fish was developed by using two sets of primers designed on a highly conserved region of the coat protein gene encoded by RNA2 of NNV. The first pair of primers amplified a fragment of 605 bp by one‐step reverse‐transcription (RT)‐PCR, while the second pair amplified an internal segment of 255 bp by nested PCR. Addition of nested PCR increased the assay sensitivity 100‐fold when carried out in a separate tube (two‐step assay) and 10‐fold when performed in the same tube (one‐step assay). The sensitivity of the two‐step assay was 104 times higher than that of virus cultivation. Nested PCR served also to confirm the specificity of the first amplification, as verified also by Southern hybridization analysis and direct sequencing. In species known to be susceptible to infection, such as European sea bass, Dicentrarchus labrax, and gilthead seabream, Sparus aurata, NNV was often detectable in brain tissue by RT‐PCR alone but only by the two‐step assay in blood, sperm, ovarian tissue or larvae. The same was true for sperm and ovarian tissue of shi drum, Umbrina cirrosa. NNV was also detected in the brains of Japanese red seabream, Pagrus major and brown meagre, Sciaena umbra, suggesting that these species can also be infected. No NNV was detected in samples of Artemia salina nauplii and rotifers obtained from a fish farm with an NNV outbreak. The inclusion of nested PCR in the assay appears to be necessary to screen out NNV‐positive broodfish by blood sampling and testing of their larval progeny.

Journal

Journal of Fish DiseasesWiley

Published: Sep 1, 2000

References

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create folders to
organize your research

Export folders, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off