Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay

Liu, Zongxiao; Teng, Yong; Liu, Hong; Jiang, Yulin; Xie, Xiayang; Li, Huifang; Lv, Jie; Gao, Longying; He, Jianping; Shi, Xiujie; Tian, Feiyan; Yang, Jingshun; Xie, Congxin

doi:10.1016/j.jviromet.2007.12.017

Cited by 48 publications

(27 citation statements)

References 22 publications

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“…No unspecific amplifications were observed with either unrelated or related viruses. Other studies have described the development of real-time RT-PCR for the diagnosis of this virus (Chico et al 2006, Liu et al 2008, Matejusova et al 2008, Cutrín et al 2009, Garver et al 2011, Jonstrup et al 2013, Pierce et al 2013), but the detection of strains from all genotypes was not always ensured, or the procedure was not tested on a significant number of strains from each genotype. Two studies reported the testing of a procedure on a number of strains similar to that in the present study.…”

Section: Discussionmentioning

confidence: 99%

Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards

López-Vázquez¹,

Bandı́n²,

Dopazo³

2015

Dis. Aquat. Org.

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In the present study, 2 systems of real-time RT-PCR -one based on SYBR Green and the other on TaqMan -were designed to detect strains from any genotype of viral haemorrhagic septicaemia virus (VHSV), with high sensitivity and repeatability/reproducibility. In addition, the method was optimized for quantitative purposes (qRT-PCR), and standard curves with different types of reference templates were constructed and compared. Specificity was tested against 26 isolates from 4 genotypes. The sensitivity of the procedures was first tested against cell culture isolation, obtaining a limit of detection (LD) of 10 0 TCID 50 ml −1 (100-fold below the LD using cell culture), at a threshold cycle value (Ct) of 36. Sensitivity was also evaluated using RNA from crude (LD = 1 fg; 160 genome copies) and purified virus (100 ag; 16 copies), plasmid DNA (2 copies) and RNA transcript (15 copies). No differences between both chemistries were observed in sensitivity and dynamic range. To evaluate repeatability and reproducibility, all experiments were performed in triplicate and on 3 different days, by workers with different levels of experience, obtaining Ct values with coefficients of variation always < 5. This fact, together with the high efficiency and R 2 values of the standard curves, encouraged us to analyse the reliability of the method for viral quantification. The results not only demonstrated that the procedure can be used for detection, identification and quantification of this virus, but also demonstrated a clear correlation between the regression lines obtained with different standards, which will help scientists to compare sensitivity results between different studies.

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Section: Discussionmentioning

confidence: 99%

Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards

López-Vázquez¹,

Bandı́n²,

Dopazo³

2015

Dis. Aquat. Org.

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“…More recently, diagnostic assays based on real-time PCR technology have become popular for pathogen surveillance because real-time PCR assays which utilize a hydrolysis probe yield rapid results, are typically highly sensitive and specific, may provide an estimate of viral load, and lend themselves to high sample through-put (Purcell et al 2011). Several reverse transcriptase quantitative or real-time PCR (RT-qPCR or RT-rPCR) assays for IHNV have been reported previously (Overturf et al 2001, Purcell et al 2006, Dhar et al 2008, Liu et al 2008.…”

Section: Introductionmentioning

confidence: 99%

Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

Purcell¹,

Thompson²,

Garver³

et al. 2013

Dis. Aquat. Org.

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Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

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“…IPN ve IHN'nin teşhisinde virus izolasyounu, enzyme linked immunosorbent assay (ELISA) ve moleküler teknikler gibi pek çok metot kullanılmaktadır (21,28) Bugün için IPN ve IHN'nin varlığı Amerika, Avrupa, Avustralya ve Uzakdoğu da bir çok ülkede bildirilmiştir (9,11,17,25).…”

Section: Introductionunclassified

Gökkuşağı alabalıklarında (Oncorhynchus mykiss Walbaum, 1792) infeksiyöz pankreatik nekrozis ve infeksiyöz hematopoietik nekrozis virus enfeksiyonlarının varlığının araştırılması

Albayrak¹,

Özan²

2010

Ankara Üniversitesi Veteriner Fakültesi Dergisi

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Gümüşhane) tatlı sulardaki ticari gökkuşağı alabalık çiftliklerinde IPN ve IHN viruslarının varlığı araştırıldı. Bu amaçla, 2006Bu amaçla, -2007 yılları Aralık-Mart aylarında (su sıcaklığının 12 °C'nin altına düştüğü dönem) bölgedeki 32 çiftlikten, 168'i (200-500 g) porsiyon balıklardan, 61'i (305 adet yavru alabalıktan, 3-10 g'lık yavru) yavru balıklardan olmak üzere toplam 229 izolasyon materyali temin edildi. İzolasyon materyallerinin BF-2 hücresinde iki pasajı yapıldı. Cytopathic effect (CPE) gösteren hücre kültür süpernatantları antijen enzyme linked immunosorbet assay (Ag-ELISA), izolasyon materyalleri ise reverse transcriptase polymerase chain reaction (RT-PCR) ile test edildi. Sonuç olarak, Ordu, Samsun, Tokat ve Trabzon illerinde bulunan 10 çiftlikte yavru ve porsiyon balıklardan IPN virusu tespit edilirken, IHN virusu tespit edilemedi. Hücre kültürü izolasyonu ve RT-PCR testlerinin birbiriyle uyumlu olduğu ortaya konuldu.Anahtar sözcükler: CPE, infeksiyöz hematopoetik nekrozis, infeksiyöz pankreatik nekrozis, RT-PCR Investigating the presence of infectious haematopoietic necrosis and infectious pancreatic necrosis virus infections in rainbow trout (Oncorhynchus mykiss Walbaum, 1792)Summary: In this study, presence of infectious pancreatic necrosis and infectious haematopoietic necrosis (IHN) viruses were investigated in fresh water in commercial rainbow trout in middle and east Black Sea region (Samsun, Sinop, Ordu, Giresun, Trabzon, Rize, Amasya, Tokat, Gümüşhane). For this purpose, total of 229 isolation material from 168 portion rainbow trout of various sizes (200 to 500 g) and 61 immature fish (from 305 immature fish, weighing 3-10 g) were obtained from 32 farms in the region in December and March in 2006-2007 which period is water temperature under the 12 °C. All isolation materials were passaged in BF-2 (Bluegill fry-2) cell cultures for two times. While cell culture supernatants showed cytopathic effect (CPE) were tested by antigen-capture enzyme linked immunosorbent assay (Ag-ELISA), all isolation materials were tested by reverse transcriptase polymerase cahin reaction (RT-PCR). As a result, while IPN virus was detected in immature fish and portion rainbow trout in 10 farms in Ordu, Samsun, Tokat and Trabzon provinces, IHN virus was not detected. That cell culture isolation and RT-PCR test are harmonious were revealed. Key words: CPE, infectious haematopoietic necrosis, infectious pancreatic necrosis, RT-PCR. GirişTürkiye'de son yıllarda hızla gelişmekte olan kültür balıkçılığı, Karadeniz Bölgesi'nde de pek çok alabalık üretim çiftliğinin kurulmasına neden olmuştur. Ülkemizde su ürünleri yetiştiriciliği yapan işletmelerin yaklaşık olarak % 70'ini alabalık çiftlikleri oluşturmaktadır (1,2).İnfeksiyöz pankreatik nekrozis (IPN) genç salmonidlerin oldukça bulaşıcı, pankreas nekrozu ile karakterize, perakut ve akut seyirli, mortalitesi yüksek viral bir enfeksiyonudur (8,12,25). İnfeksiyöz hematopoetik nekrozis (IHN) ise, alabalıklarda hematopoetik dokuların nekrozu ile karakterize akut ve subakut s...

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Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay

Cited by 48 publications

References 22 publications

Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards

Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards

Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

Gökkuşağı alabalıklarında (Oncorhynchus mykiss Walbaum, 1792) infeksiyöz pankreatik nekrozis ve infeksiyöz hematopoietik nekrozis virus enfeksiyonlarının varlığının araştırılması

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