Rapid and sensitive detection of shrimp yellow head virus using loop-mediated isothermal amplification and a colorogenic nanogold hybridization probe

Aims: Enterocytozoon hepatopenaei is an emerging microsporidian parasite that has been linked to recent losses caused by white faeces syndrome (WFS) in cultivated giant or black tiger shrimp Penaeus (Penaeus) monodon and whiteleg shrimp Penaeus (Litopenaeus) vannamei in Asia. To more accurately assess its impact on shrimp production and to determine reservoir carriers for control measures, our objective was to establish a loop-mediated isothermal amplification (LAMP) assay combined with colorimetric nanogold (AuNP) for rapid, sensitive and inexpensive detection of this parasite. Methods and Results: A set of six specific primers was designed to successfully detect the SSU rRNA gene of E. hepatopenaei by a LAMP reaction of 45 min at 65°C combined with visual detection of the amplification product via hybridization at 65°C for 5 min with a ssDNA-labelled nanogold probe, followed by salt-induced AuNP aggregation (total assay time, approximately 50 min). This method gave similar results to LAMP followed by electrophoresis or spectrophotometric detection, and it was more sensitive (0Á02 fg total DNA) than a conventional nested PCR (0Á2 fg total DNA). The new method gave negative results with shrimp DNA templates extracted from diseased shrimp containing other pathogens, indicating that the LAMP-AuNP assay was specific for E. hepatopenaei. Conclusions: Without sacrificing sensitivity or specificity, the new LAMPAuNP assay significantly reduced the time, ease and cost for molecular detection of E. hepatopenaei in shrimp. Significance and Impact of the study: The new method employs simple, inexpensive equipment and involves simple steps making it applicable for small field laboratories. Wider application of the method to screen broodstock before use in a hatchery, to screen postlarvae before stocking shrimp ponds, to test for natural carriers and to monitor shrimp in rearing ponds would help to assess and reduce the negative impact of this parasite in shrimp farming.

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“…The ssDNA-labelled AuNP probe was prepared by modification of a previous method (Jaroenram et al 2012). In brief, 2 and 5 nmol l…”

Section: Gold Nanoparticles (Aunp) Probe Preparationmentioning

confidence: 99%

Retracted: Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidianEnterocytozoon hepatopenaeiin penaeid shrimp

Suebsing

Prombun

Srisala

et al. 2013

Journal of Applied Microbiology

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“…10,11 These methods can allow for real-time monitoring of signals but sacrifice specificity because any accumulation of non-specific or parasitic amplicons will yield a false positive signal. While some sequence-specific detection methods have also been developed, such as fluorescent resonance transfer (FRET) probes, 12,13 molecular zippers, 14 molecular beacons, 15,16 and hybridization between DNA-coated gold nanoparticles, 17,18 these approaches merely mirror the development of amplicons and provide no opportunity for programming how amplicon signals might be amplified, integrated, and displayed in real-time.…”

mentioning

confidence: 99%

Real-Time Detection of Isothermal Amplification Reactions with Thermostable Catalytic Hairpin Assembly

et al. 2013

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Catalytic hairpin assembly (CHA) is an enzyme-free amplification method that has previously proven useful in amplifying and transducing signals at the terminus of nucleic acid amplification reactions. Here, for the first time, we engineered CHA be thermostable from 37 °C to 60 °C and in consequence have generalized its application to real-time detection of isothermal amplification reactions. CHA circuits were designed and optimized for both high and low temperature rolling circle amplification (RCA) and strand displacement amplification (SDA). The resulting circuits not only increased the specificity of detection, they also improved sensitivity by as much as 25- to 10,000-fold over comparable real-time detection methods. These methods have been condensed into a set of general rules for the design of thermostable CHA circuits with high signals and low noise.

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“…(2) For arthropod: Chikungunya viruses [11] and arthropodborne West Nile viruses [69]. (3) For fish and other marine animals: koi herpes viruses [19,70,71], lymphocytis disease viruses for fish [54], infectious hematopoietic necrosis viruses (IHNV) in rain-bow trout [72], viral hemorrphagic septicemia viruses [73], infectious spleen and kidney necrosis viruses (ISKNV) in fishes [14,74], cyprinid herpesvirus 2 (CyHV-2) [75,76], cyprinid herpesvirus 3 [77,78], Edwardsiella tarda in fish [79], Francisella piscicida in Atlantic cod [80], iridovirus in fish [36,81,82], white spot syndrome virus in shrimp [44,[83][84][85], shrimp yellow head virus (YHV) [86,87], Taura syndrome virus (TSV) in shrimp [88], shrimp infectious myonecrosis virus (IMNV) [89,90], infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp [91,92], Penaeus monodon nucleopolyhedrovirus (PemoNPV) for shrimp [93], macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) in prawn [94], acute viral necrobiotic virus in scallop [95], softshelled turtle iridovirus [96], abalone herpesviruses [39], mud crab dicistrovirus-1 [27], and ostreid herpesvirus 1 (OsHV-1) DNA [97]. (4) For birds and chicken: subgroup A and subgroup J of avian leukosis viruses [35,…”

Section: Applications Of Lampmentioning

confidence: 99%

“…Colorimetric reverse transcription (RT)-LAMP-AuNP assay is also reported for detecting shrimp yellow head virus (YHV) and shrimp infectious myonecrosis virus (IMNV) through RNA sensing [86,89]. In the presence of RT-LAMP target DNAs, the ssDNA labeled AuNP probes will still remain dispersed showing a pink color solution despite the addition of salt, while the absence of the RNA target, ssDNA-AuNPs will start to aggregate and turn into purplish solution.…”

Section: Aunp Combined With Lampmentioning

confidence: 99%

Colorimetric biosensors for point-of-care virus detections

Zhao

Wong

Zheng

et al. 2020

Materials Science for Energy Technologies

102

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a b s t r a c tColorimetric biosensors can be used to detect a particular analyte through color changes easily by naked eyes or simple portable optical detectors for quantitative measurement. Thus, it is highly attractive for point-of-care detections of harmful viruses to prevent potential pandemic outbreak, as antiviral medication must be administered in a timely fashion. This review paper summaries existing and emerging techniques that can be employed to detect viruses through colorimetric assay design with detailed discussion of their sensing principles, performances as well as pros and cons, with an aim to provide guideline on the selection of suitable colorimetric biosensors for detecting different species of viruses. Among the colorimetric methods for virus detections, loop-mediated isothermal amplification (LAMP) method is more favourable for its faster detection, high efficiency, cheaper cost, and more reliable with high reproducible assay results. Nanoparticle-based colorimetric biosensors, on the other hand, are most suitable to be fabricated into lateral flow or lab-on-a-chip devices, and can be coupled with LAMP or portable PCR systems for highly sensitive on-site detection of viruses, which is very critical for early diagnosis of virus infections and to prevent outbreak in a swift and controlled manner.

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Rapid and sensitive detection of shrimp yellow head virus using loop-mediated isothermal amplification and a colorogenic nanogold hybridization probe

Cited by 49 publications

References 33 publications

Retracted: Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidianEnterocytozoon hepatopenaeiin penaeid shrimp

Retracted: Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidianEnterocytozoon hepatopenaeiin penaeid shrimp

Real-Time Detection of Isothermal Amplification Reactions with Thermostable Catalytic Hairpin Assembly

Colorimetric biosensors for point-of-care virus detections

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