Improvement of a PCR Method with the Sph I-5 Primer Set for the Detection of Koi Herpesvirus (KHV)

Yuasa, Kei; Sano, Motohiko; Kurita, Jun; Ito, Takafumi; Iida, Takaji

doi:10.3147/jsfp.40.37

Cited by 61 publications

(58 citation statements)

References 1 publication

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…RNA extracts were treated by incolumn digestion with DNase I (Qiagen) according to the manufacturer's instructions. Two PCR assays (Bercovier et al 2005, Yuasa et al 2005 were performed with 1 µl of each DNase-treated RNA extract to confirm removal of DNA.…”

Section: Preliminary Test Dnase Treatment To Remove Dna From Samplesmentioning

confidence: 99%

“…Extraction protocols used for genomic DNA and RNA were the same as those employed for the cultured cells (Test 1). Amplification of the genomic KHV DNA was performed according to Yuasa et al (2005). RT-PCR for detecting the mRNA was conducted with primer set A using the optimal thermal protocol (RT at 55°C for 30 min, with inactivation of reverse transcriptase at 94°C for 2 min followed by 35 cycles of denaturation at 94°C at 30 s, annealing at 60°C for 30 s, and elongation at 72°C for 30 s followed by a final elongation step of 72°C for 7 min).…”

Section: Verification Test 5 Detection Of Genomic Dna and Terminase mentioning

confidence: 99%

“…PCR products were not obtained using the PCR assays of Yuasa et al (2005) and Bercovier et al (2005) with DNAse-treated gill RNA extracted at 3 dpi. However, both PCR assays amplified a product of 292 or 409 bp, respectively, from DNase-treated RNA template extracted from gills sampled at 7 dpi (Fig.…”

Section: Preliminary Test Dnase Treatment To Remove Dna From Samplesmentioning

confidence: 99%

“…All PCR methods that have been developed to date to detect cyprinid herpesvirus 3 (CyHV-3, Waltzek et al 2005), commonly known as koi herpesvirus (KHV), are highly sensitive and specific for the virus (Gilad et al 2002, Gray et al 2002, Bercovier et al 2005, Yuasa et al 2005. Given the relative resilience of DNA to degradation, PCR assays of KHV DNA extracted from viral capsids are generally successful, even when using tissues that have started to decompose (authors' unpubl.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Development of mRNA-specific RT-PCR for the detection of koi herpesvirus (KHV) replication stage

Yuasa¹,

Kurita²,

Kawana³

et al. 2012

Dis. Aquat. Org.

View full text Add to dashboard Cite

An mRNA-specific reverse transcription (RT)-PCR primer set spanning the exon junction of a spliced putative terminase gene in the koi herpesvirus (KHV) was developed to detect the replicating stage of the virus. The proposed RT-PCR amplified a target gene from the RNA template, but not from a DNA template extracted from common carp brain (CCB) cells infected with KHV. In addition, the RT-PCR did not amplify the target gene of templates extracted from specific cell lines infected with either CyHV-1 or CyHV-2. RT-PCR detected mRNA from the scales of koi experimentally infected with KHV at 24 h post exposure (hpe). However, unlike conventional PCR, RT-PCR could not detect KHV DNA in fish at 0 hpe. The results indicate that the RT-PCR developed in this study is mRNA-specific and that the assay can detect the replicating stage of KHV from both fish and cultured cells infected with the virus.

show abstract

Section: Preliminary Test Dnase Treatment To Remove Dna From Samplesmentioning

confidence: 99%

Section: Verification Test 5 Detection Of Genomic Dna and Terminase mentioning

confidence: 99%

Section: Preliminary Test Dnase Treatment To Remove Dna From Samplesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Development of mRNA-specific RT-PCR for the detection of koi herpesvirus (KHV) replication stage

Yuasa¹,

Kurita²,

Kawana³

et al. 2012

Dis. Aquat. Org.

View full text Add to dashboard Cite

show abstract

“…Several diagnostic tests based on PCR [6,25,29,89], nested PCR [7,20], real-time PCR [27] and loop-mediated isothermal amplification (LAMP) [31,75,87,88] have been developed for detection of KHV. A highly sensitive PCR method for detection of TK gene of KHV is able to detect 10 fg of KHV DNA [6].…”

Section: Molecular Detection Of Viral Dnamentioning

confidence: 99%

Koi Herpes Virus: A Review and Risk Assessment of Indian Aquaculture

et al. 2012

View full text Add to dashboard Cite

Common carp (Cyprinus carpio) is a widely cultivated freshwater fish for human consumption, while koi carp, is a farmed colored sub species of common carp used for ornamental purposes. Since 1998, both common carp and koi carp are severely affected by a viral disease called as Koi herpes virus disease (KHVD). This disease is caused by Koi herpes virus (KHV), also known as cyprinid herpes virus-3. The virus causes interstitial nephritis and gill necrosis in carps, so it is also termed as carp interstitial nephritis and gill necrosis virus. KHV is a double stranded icosahedral DNA virus belonging to family Alloherpesviridae, with a genome size of 295 kbp, larger than any member of Herpesviridae. The viral genome encodes 156 potential protein coding open reading frames. Each virion consists of forty structural proteins, which are classified as capsid (3), envelope (13), tegument (2) and unclassified (22) structural proteins. Diagnosis of KHVD is mainly based on detection of viral DNA by polymerase chain reaction amplification using specific primers or loop mediated isothermal amplification. Temperature dependent latent infection is unique to KHV; and carrier fish are often not detected, thereby possibly resulting in spread of this pathogen to newer areas. The disease is now known to occur in, or has been recorded from at least 26 different countries of the world. Fortunately, KHVD has not been reported from India or from Indian major carps. To monitor the disease status of the country, a total of 254 fish samples collected from different parts of India were screened by PCR for the presence of KHV. None of the tested samples were found to be positive for KHV. These results demonstrate that tested samples from different parts of India were apparently free from KHV. Preliminary risk assessment of KHV suggest that in the event of unrestricted importation of koi carps into our country, there is a higher probability of risk to aquaculture as compared to natural waters. So there is strong need to develop diagnostic capabilities and launch surveillance programmes for KHV in India.

show abstract

Overview of Fish Immune System and Infectious Diseases

Shoemaker

LaFrentz

et al. 2015

Dietary Nutrients, Additives, and Fish Health

View full text Add to dashboard Cite

Improvement of a PCR Method with the Sph I-5 Primer Set for the Detection of Koi Herpesvirus (KHV)

Cited by 61 publications

References 1 publication

Development of mRNA-specific RT-PCR for the detection of koi herpesvirus (KHV) replication stage

Development of mRNA-specific RT-PCR for the detection of koi herpesvirus (KHV) replication stage

Koi Herpes Virus: A Review and Risk Assessment of Indian Aquaculture

Overview of Fish Immune System and Infectious Diseases

Contact Info

Product

Resources

About