Wansika Kiatpathomchai

Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions using a set of four specifically designed primers that recognize six distinct target sequences. It can be combined with a chromatographic lateral flow dipstick (LFD) for highly specific, rapid and simple visual detection of WSSV-specific amplicons. Using this protocol, a 30-min amplification followed by 5 min hybridization with an FITC-labeled DNA probe and 5 min LFD resulted in visualization of DNA amplicons trapped at the LFD test line. Thus, 10 min for rapid DNA extraction followed by LAMP combined with LFD detection resulted in a total assay time of approximately 50 min. Detection sensitivity was comparable to other commonly-used methods for nested PCR detection of WSSV but had the additional advantages of reduced assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide.

Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acid under isothermal conditions using four sets of specially designed primers that recognize six distinct target sequences with high specificity and sensitivity. In this report, a 60-min reverse transcription LAMP (RT-LAMP) method for amplification of Taura syndrome virus (TSV) cDNA using biotin-labeled primer was combined with a chromatographic lateral flow dipstick (LFD) for rapid and simple visual detection of TSV-specific amplicons. The LFD process involved a 5-min post RT-LAMP step for specific hybridization of cDNA with an FITC-labeled DNA probe that confirmed the presence of specific, biotin-labeled TSV amplicons. The resulting DNA duplexes could be visualized trapped at the LFD strip test line within 5min of sample exposure. Using the combined RT-LAMP and LFD system, the total assay interval was approximately 70min, excluding RNA extraction time. Detection sensitivity was comparable to other commonly used methods for nested RT-PCR detection of TSV. In addition to reduced assay time when compared to electrophoresis, combination of RT-LAMP with LFD confirms amplicon identity by hybridization and eliminates the need to handle carcinogenic ethidium bromide.

Detection of shrimp infectious myonecrosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.

Infectious myonecrosis virus (IMNV) has caused a slowly progressive disease with cumulative mortalities of up to 70% or more in cultured Penaeus (Litopenaeus) vannamei in Northeast Brazil and Indonesia. Rapid detection of viruses by loop-mediated isothermal amplification (LAMP) of genomic material with high specificity and sensitivity can be applied for diagnosis, monitoring and control of diseases in shrimp aquaculture. Using an IMNV template, successful detection was achieved after a 60-min RT-LAMP reaction using biotin-labeled primers followed by 5min hybridization with an FITC-labeled DNA probe and 5min assay using a chromatographic lateral flow dipstick (LFD). Thus, the combined system of RT-LAMP and LFD required a total assay interval of less than 75min, excluding the RNA extraction time. The sensitivity of detection was comparable to that of other commonly used methods for nested RT-PCR detection of IMNV. In addition to reducing amplicon detection time when compared to electrophoresis, LFD confirmed amplicon identity by hybridization and eliminated the need to handle carcinogenic ethidium bromide. The RT-LAMP-LFD method gave negative test results with nucleic acid extracts from normal shrimp and from shrimp infected with other viruses including infectious hypodermal hematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), a hepatopancreatic parvovirus from P. monodon (PmDNV), white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Macrobrachium rosenbergii nodavirus (MrNV) and gill associated virus (GAV).

Shrimp hepatopancreatic parvovirus detection by combining loop-mediated isothermal amplification with a lateral flow dipstick

Present methods such as traditional PCR, PCR-ELISA, real-time PCR and histopathology for detection of shrimp hepatopancreatic parvovirus (PmDNV) entail various disadvantages including high cost, long assay time or use of toxic substances. Loop-mediated isothermal amplification (LAMP) of target nucleotide sequences under inexpensive isothermal conditions combined with amplicon detection by chromatographic lateral flow dipsticks (LFD) allowed simpler detection within 75 min. Biotinylated LAMP amplicons from the targeted portion of the PmDNA capsid protein gene were produced under isothermal conditions at 63 degrees C for 1h and then hybridized at 63 degrees C for 5 min with an FITC-labeled probe (optimized at 20 pmol) that was specific for the LAMP amplicons (i.e., outside the primer region). The FITC-labeled, biotinylated LAMP product picked up gold-labeled, anti-FITC near the LFD origin and the whole, triple-labeled complex was captured by an immobilized biotin-binding protein to yield a red nano-gold stripe at the LFD test line. With a DNA template derived from PmDNV-infected shrimp, the LAMP-LFD detection limit was 1 ng while that for one-step PCR-electrophoresis was 10 ng. Comparative sensitivity for one nested-PCR-electrophoresis method was 1 ng but for another 0.1 ng. The LAMP-LFD method gave negative test results with DNA extracts from normal shrimp and from shrimp infected with other DNA viruses including monodon baculovirus (MBV), white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV).

Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) by loop-mediated isothermal amplification combined with a lateral-flow dipstick

Several methods such as traditional PCR or nested-PCR, immuno assay and histopathology have been developed for detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) formerly called monodon baculovirus (MBV). However, these methods have various disadvantages including low sensitivity, long assay time, use of toxic substances or unsuitability for field diagnosis. Loop-mediated isothermal amplification of target nucleotide sequences under isothermal conditions, combined with amplicon detection by chromatographic lateral-flow dipsticks allows for more efficient, field friendly detection within 75 min (not including DNA preparation time). In this study, the LAMP amplicon was biotinylated via an inner LAMP primer designed from a BamHI fragment B, a hypothetical protein gene of PemoNPV under isothermal condition at 63 degrees C for 1 h. Next, the LAMP product was hybridized at 63 degrees C for 5 min with an optimal FITC-labeled probe that was designed specifically for the LAMP amplicons. The FITC-labeled biotinylated LAMP product picked up gold-labeled, anti-FITC near the LFD origin and the whole, triple-labeled complex was captured by an immobilized biotin-binding protein to yield a red nano-gold stripe at the LFD test line. With a DNA template extracted from PemoNPV-infected shrimp, the LAMP-LFD detection limit was 0.1 pg, whereas one-step PCR and nested-PCR followed with gel electrophoresis was 1 pg. The LAMP-LFD method gave negative test results with buffer and DNA from shrimp infected with other common shrimp DNA viruses including, Penaeus monodon densovirus (PmDNV) formerly called hepatopancreatic parvovirus (HPV), white spot syndrome virus (WSSV) and Penaeus stylirostris densovirus (PstDNV) formerly called infectious hypodermal and hematopoietic necrosis virus (IHHNV). The test platform can be adapted easily for rapid detection of other shrimp viruses, since the LAMP-LFD combination system was a highly sensitive, specific, convenient, and does not require sophisticated instruments.

Visual detection of white spot syndrome virus using DNA-functionalized gold nanoparticles as probes combined with loop-mediated isothermal amplification

The integration of loop-mediated isothermal amplification (LAMP) and DNA-functionalized AuNPs as visual detection probes (LAMP-AuNPs) was developed and applied for the detection of white spot syndrome virus (WSSV) from Penaeid shrimp in this study. The principle of this combination assay relies on the basis of stability characteristics of the DNA-functionalized AuNPs upon hybridization with the complementary target DNA toward salt-induced aggregation. If the detected target DNA is not complementary to the ssDNA probes, the DNA-functionalized AuNPs will be aggregated due to the screening effect of salt, resulting in the change of solution color from red to blue/gray and shift of the surface plasmon peak to longer wavelength. While the DNA-functionalized AuNPs are perfectly matched to the detected target DNA, the color of solution still remains red in color and no surface plasmon spectral shift. This assay provides simply technique, time-saving and its detection results could be achieved qualitatively and quantitatively by visualization using the naked eye due to the colorimetric change and by measurement using the UV-vis spectroscopy due to the surface plasmon spectral shift, respectively. In this study, LAMP-AuNPs assay was successfully developed with the detection of WSSV-LAMP generated product at 0.03 μg/reaction, and showed the sensitivity of 2 × 10(2) copies WSSV plasmid DNA, that is comparable to the most sensitive method reported to date. The LAMP-AuNPs assay described in this study revealed a highly sensitive, rapid and reliable diagnostic protocol for detection of WSSV. This technique has a potential as a routine method for assessing the infectious diseases in Penaeid shrimp not only for WSSV, but also for other shrimp pathogens, and can be useful tool in field conditions for the diagnosis or surveillance programs.

Rapid and sensitive detection of infectious hypodermal and hematopoietic necrosis virus by loop-mediated isothermal amplification combined with a lateral flow dipstick

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is an important shrimp pathogen that causes mortality in Penaeus stylirostris and stunting (called runt deformity syndrome or RDS) in Penaeus vannamei. Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. It can be combined with a chromatographic lateral flow dipstick (LFD) for highly specific, rapid and simple visual detection of IHHNV-specific amplicons. Using this protocol, a 30-min amplification followed by 5 min hybridization with an FITC-labeled DNA probe and 5 min LFD resulted in visualization of DNA amplicons trapped at the LFD test line. Thus, 10 min for rapid DNA extraction followed by LAMP combined with LFD detection resulted in a total assay time of approximately 50 min. Detection sensitivity was comparable to other methods used commonly for nested PCR detection of IHHNV but had the additional advantages of reduced assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide.

Rapid and sensitive detection of Macrobrachium rosenbergii nodavirus in giant freshwater prawns by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. It can be combined with a chromatographic lateral flow dipstick (LFD) for much more efficient, field-friendly detection of MrNV. In this work, RT-LAMP was performed at 65 degrees C for 40 min, followed by 5 min for hybridization with an FITC-labeled DNA probe and 5 min for LFD resulted in visualization of DNA amplicons trapped at the LFD test line. Thus, total assay time, including 10 min for rapid RNA extraction was approximately 60 min. In addition to advantages of short assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide, the RT-LAMP-LFD was more sensitive than an existing RT-PCR method for detection of MrNV. The RT-LAMP-LFD method gave negative test results with nucleic acid extracts from normal shrimp and from shrimp infected with other viruses including DNA viruses [PstDNV (IHHNV), PemoNPV (MBV), PmDNV (HPV), WSSV] and RNA viruses (TSV, IMNV, YHV/GAV).

Application of loop-mediated isothermal amplification assay combined with lateral flow dipstick for detection of Plasmodium falciparum and Plasmodium vivax

Malaria is largely a preventable and curable disease. However, a delay or an inappropriate treatment can result in serious adverse outcomes for patient. Rapid, simple and cost-effective diagnostic tests that can be easily adapted and rapidly scaled-up at the field or community levels are needed. In this study, accelerated detection methods for the Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) dihydrofolate reductase-thymidylate synthase were developed based on the loop-mediated isothermal amplification (LAMP) method. The developed methods included the use of species-specific biotinylated primers to amplify LAMP amplicons, which were then hybridized to specific FITC-labeled DNA probes and visualized on a chromatographic lateral flow dipstick (LFD). The total LAMP-LFD assay time was approximately 1.5h. The LAMP-LFD assays showed similar detection limit to conventional PCR assay when performed on plasmid DNA carrying the malaria dhfr-ts genes. The LAMP-LFD showed 10 folds higher detection limit than PCR when performed on genomic DNA samples from Pf and Pv parasites. The dhfr-ts LAMP-LFD assays also have the advantages of reduced assay time and easy format for interpretation of results.

Loop‐mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp

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.