A loop mediated isothermal amplification (LAMP) method for detection of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss)

Gunimaladevi, I.; Kono, Tomoya; LaPatra, Scott E.; Sakai, Masahiro

doi:10.1007/s00705-004-0468-7

Cited by 64 publications

(40 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, although considered rapid and specific methods for detection, these require expensive equipment and other costly materials (Gunimaladevi et al 2005).…”

Section: Resale or Republication Not Permitted Without Written Consenmentioning

confidence: 99%

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Tsai¹,

Wang²,

Yoshida³

et al. 2013

Dis. Aquat. Org.

View full text Add to dashboard Cite

Based on use of a loop-mediated isothermal amplification (LAMP) identification protocol, this study attempted to detect Lactococcus garvieae in fish by using primer sets designed from an L. garvieae alpha/beta fold family hydrolase gene. Reaction time and temperatures were optimized for 60 min at 60°C with the resulting amplicons visualized by adding SYBR Green I to the reaction tube. The assay specificity was assessed using 45 different bacterial strains. Positive results were observed in all 30 L. garvieae isolates from various aquatic animals. No false-positive results were observed in 15 non-L. garvieae strains. The detection limit of the LAMP assay was 10-fold more sensitive than the conventional polymerase chain reaction (PCR) targeting 16S rDNA when using purified L. garvieae DNA. The detection limit of the LAMP assay was approximately 300 colony-forming units (CFU) using crude bacterial lysates, 100-fold more sensitive than PCR. Furthermore, L. garvieae in spleen, kidney and brain of experimentally challenged tilapia and grey mullet were detected using this optimized LAMP assay. Results of this study demonstrate the effectiveness of LAMP in providing a rapid yet simple test for detecting L. garvieae in fish.KEY WORDS: Lactococcosis · Alpha/beta fold family · Hydrolase gene · Loop-mediated isothermal amplification · Polymerase chain reaction · SYBR Green I Resale or republication not permitted without written consent of the publisherDis Aquat Org 102: [225][226][227][228][229][230][231][232][233][234][235] 2013 swimming. However, similar clinical signs are also found in Streptococcus sp. infections (Eldar & Ghittino 1999).Molecular detection methods of Lactococcus garvieae have been developed, including polymerase chain reaction (PCR) (Zlotkin et al. 1998, Aoki et al. 2000, Pu et al. 2002 and 16S rRNA and sodA sequence analysis (Fihman et al. 2006). However, although considered rapid and specific methods for detection, these require expensive equipment and other costly materials (Gunimaladevi et al. 2005).A method of amplifying nucleic acid sequences with high specificity, sensitivity and rapidity under isothermal conditions (60 to 65°C), loop-mediated isothermal amplification (LAMP) has been developed more recently (Notomi et al. 2000) and applied to fish pathogen detection (Caipang et al. 2004, Savan et al. 2004, Yeh et al. 2005, Itano et al. 2006. The assay uses a set of 4 or 6 primers that create a targetspecific stem loop DNA structure during initial amplification steps; subsequent LAMP auto-cycling is performed by the Bst DNA polymerase large fragment (Notomi et al. 2000). Since it does not require complicated equipment, the LAMP method provides a cost-effective, simple, and rapid (producing a result within 60 min) DNA amplification method.Recently, a fosmid library of Lactococcus garvieae from grey mullet has been constructed and many new genes of L. garvieae were cloned (M. A. Tsai et al. unpubl. data). The clone flg08-6 contained an alpha/beta fold family hydrolase gene that is also...

show abstract

“…However, although considered rapid and specific methods for detection, these require expensive equipment and other costly materials (Gunimaladevi et al 2005).…”

Section: Resale or Republication Not Permitted Without Written Consenmentioning

confidence: 99%

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Tsai¹,

Wang²,

Yoshida³

et al. 2013

Dis. Aquat. Org.

View full text Add to dashboard Cite

show abstract

“…Specificity and efficiency was found be highly dependent on Mg 2+ concentration. Mg 2+ is known to affect primer annealing (Nie 2005;Gunimaladevi et al 2005;Teng et al 2007;Liu et al 2010) and it is possible that the LAMP conditions make those conditions too stringent for primers to anneal to the slight variations in sequence. RT-LAMP ideally should be able to detect all strains of the GLRaV-3 and as such it is important that the conditions not be too stringent.…”

Section: Discussionmentioning

confidence: 99%

Rapid detection of Grapevine leafroll-associated virus type 3 using a reverse transcription loop-mediated amplification method

Walsh

Pietersen

2013

Journal of Virological Methods

View full text Add to dashboard Cite

“…LAMP is a novel method of DNA amplification that has already been applied to detection of several pathogens in aquaculture (Savan et al 2005). These include IHNV (Gunimaladevi et al 2005, McCarthy et al 2006, infectious salmon anemia virus (ISAV) (McCarthy et al 2006), VHSV (Soliman & ElMatbouli 2006), spring viraemia of carp virus (SVCV) (Liu et al 2008), IPNV (Soliman et al 2009), turbot reddish body iridovirus (TRBIV) (Zhang et al 2009), white spot syndrome virus (WSSV) , koi herpesvirus (KHV) (Gunimaladevi et al 2004), yellow head virus (YHV) (Mekata et al 2006) and Taura syndrome virus (TSV) (Kiatpathomchai et al 2007). According to these studies, the sensitivity of the LAMP assay is higher than the conventional PCR assay.…”

Section: Discussionmentioning

confidence: 99%

“…With betaine, the amplification of short target fragments (< 300 bp) was not affected, but non-specific amplification, as reported by Baskaran et al (1996), was reduced. The levels of Bst DNA polymerase (6 U) and AMV reverse transcriptase (0.25 U) were also optimized for use under optimal MgSO 4, betaine and dNTPs concentrations.The RT-LAMP technique for detecting IHNV was previously reported in Japan (Gunimaladevi et al 2005). In their experiment, IHNV-specific LAMP ) and there is no report on the occurrence of the M genogroup in Korea at the present time.…”

mentioning

confidence: 93%

Reverse transcriptase loop-mediated isothermal amplification assay for infectious hematopoietic necrosis virus in Oncorhynchus keta

Suebsing¹,

Jeon²,

Oh³

et al. 2011

Dis. Aquat. Org.

View full text Add to dashboard Cite

A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting infectious hematopoietic necrosis virus (IHNV) from chum salmon Oncorhynchus keta in South Korea with high specificity, sensitivity and rapidity. A set of 6 IHNV-specific primers was designed, based on the G-protein sequence of IHNV (PRT strain), recognizing 8 distinct sequences of the target RNA. The assay was optimized to detect IHNV at 63°C for 30 min. The limit of detection was 0.01 fg of RNA extracted from IHNV-infected CHSE-214 cells, compared with 1.0 fg for nested RT-PCR. The applicability of this RT-LAMP assay was further tested by comparison with nested RT-PCR using field samples. Of 473 samples tested, 191 samples (40.38%) were IHNVpositive by RT-LAMP, whereas 162 samples (34.25%) were IHNV-positive by nested RT-PCR. These results indicate that, because of its high sensitivity and rapidity, the RT-LAMP assay is useful for early diagnosis of IHN. KEY WORDS: RT-LAMP · IHNV · Chum salmon · Oncorhynchus keta Resale or republication not permitted without written consent of the publisherDis Aquat Org 94: [1][2][3][4][5][6][7][8] 2011 method. However, nested RT-PCR methods require a second amplification step that is prone to contamination. To solve these problems, the loop-mediated isothermal amplification (LAMP) assay was investigated as an alternative. LAMP was originally developed by Notomi et al. (2000) and can amplify very low numbers of target sequences to 10 9 copies under isothermal conditions within 1 h. This method depends on auto-cycling strand displacement DNA synthesis by the Bst DNA polymerase large fragment with high strand displacement activity, and a set of 2 specially designed inner primers and 2 outer primers. LAMP is highly specific because the target sequences are detected by 6 independent primers in the initial stage, followed by 4 independent primers in the later stages of the LAMP reaction. LAMP is also applicable for RNA detection by using a reverse transcriptase (RT) together with a DNA polymerase (Notomi et al. 2000). This technique can be carried out under isothermal conditions, which can be simply achieved with a water bath or a heating block. Expensive thermal cyclers used for PCR are not required (Notomi et al. 2000).In the present study, an RT-LAMP assay was developed for detecting IHNV from wild adult chum salmon Oncorhynchus keta and artificially hatched fry in Korea. A set of 6 LAMP-specific primers was designed, based on the G protein sequence of Korean IHNV (PRT strain, accession no. AY673684). The applicability of the RT-LAMP assay was evaluated by comparison with the nested RT-PCR assay using field samples. MATERIALS AND METHODSViruses and cell culture. IHNV (PRT strain) was amplified in chinook salmon embryo (CHSE)-214 cells. Infectious pancreatic necrosis virus (IPNV) (VR-299, Jasper, Sp and Ab strains) and viral hemorrhagic septicemia virus (VHSV) were maintained in rainbow trout gonad (RTG)-2 cells with minimum essential medium-10 (supplemented wit...

show abstract

A loop mediated isothermal amplification (LAMP) method for detection of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss)

Cited by 64 publications

References 26 publications

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Rapid detection of Grapevine leafroll-associated virus type 3 using a reverse transcription loop-mediated amplification method

Reverse transcriptase loop-mediated isothermal amplification assay for infectious hematopoietic necrosis virus in Oncorhynchus keta

Contact Info

Product

Resources

About