Transcriptase Loop-mediated Isothermal Amplification Assay Research Articles

Development and Validation of a Colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Eastern Equine Encephalitis Virus in Mosquitoes.

Eastern equine encephalitis virus (EEEV) is a highly pathogenic alphavirus that causes periodic outbreaks in the eastern USA. Mosquito abatement programs are faced with various challenges with surveillance and control of EEEV and other mosquito-borne illnesses. Environmental sampling of mosquito populations can be technically complex. Here we report the identification of biomarkers, development and validation of a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of EEEV. Positive samples are easily visualized by a color change from pink to yellow. The assay was validated using EEEV from viral culture, experimentally spiked mosquito pools, and previously tested mosquito pools. The RT-LAMP assay detected viral titers down to approximately 10% of what would be present in a single infectious mosquito, based upon EEEV viral titers determined by previous competency studies. The RT-LAMP assay efficiently detected EEEV in combined aliquots from previously homogenized pools of mosquitoes, allowing up to 250 individual mosquitoes to be tested in a single reaction. No false positive results were obtained from RNA prepared from negative mosquito pools acquired from known and potential EEEV vectors. The colorimetric RT-LAMP assay is highly accurate, technically simple, and does not require sophisticated equipment, making it a cost-effective alternative to real time reverse transcriptase-polymerase chain reaction (RT-PCR) for vector surveillance.

Immunocapture-Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Plant RNA Viruses.

Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a specific nucleic acid target with high specificity. The LAMP reaction process has no denaturation step, instead DNA amplification occurs by strand displacement activity of the Bacillus stearothermophilus (Bst) DNA polymerase under isothermal conditions. It utilizes three sets of forward and reverse oligonucleotide primers specific to six distinct sequences on the target gene. These primers are used to generate amplification products that contain single-stranded loops, thereby allowing primers to bind to these sequences without the need for repeated cycles of thermal denaturation. For diagnosis of pathogens with RNA genome, LAMP has been merged with reverse transcription (RT) step to create RT-LAMP. To further reduce the cost of diagnosis and increase the throughput, immunocapture (IC) step was added to develop IC-RT-LAMP assay. Hence, this chapter focuses on utilizing IC-RT-LAMP assay to specifically identify severe strain of a plant virus from field samples.

A rapid detection tool for VT isolates of Citrus tristeza virus by immunocapture-reverse transcriptase loop-mediated isothermal amplification assay.

Severe strains of Citrus tristeza virus (CTV) cause quick decline and stem pitting resulting in significant economic losses in citrus production. A immunocapture reverse-transcriptase loop-mediated amplification (IC-RT-LAMP) assay was developed in this study to detect the severe VT strains that are typically associated with severe CTV symptoms. The sensitivity of RT-LAMP assay was determined by ten-fold serial dilutions of CA-VT-AT39 RNA, in comparison to one-step RT-droplet digital (dd) PCR. RT-LAMP detected up to 0.002 ng RNA with an amplification time of 10:35 (min:sec.), equivalent to 11.3 copies as determined by one step RT-ddPCR. The RT-LAMP assay specifically detected CA-VT-AT39 RNA and did not cross react with other CTV genotypes tested (T36, T30, RB, S1 and T68). To facilitate rapid on-site detection, the RT-LAMP assay was improved by first capturing the CTV virions from citrus crude leaf sap using CTV-IgG (IC-RT-LAMP), thereby eliminating nucleic acid extraction steps. IC-RT-LAMP assay was optimized with two-fold dilutions of CTV-IgG ranging from 1:500 to 1:16,000. The IC-RT-LAMP assay detected the CA-VT-AT39 virions in all dilutions tested. The minimum amplification time was 6:45 (min:sec) with 1:500 and 1:1000 of CTV-IgG dilutions. The limit of detection of IC-RT-LAMP assay with crude leaf sap of CA-VT-AT39 was 1:320 with a maximum amplification time of 9:08 (min:sec). The IC-RT-LAMP assay was validated for VT genotype by comparing to IC-RT-qPCR using the CTV from 40 field tree samples. A 100% agreement was observed between tests, regardless of single or mixed infections of CTV VT with other genotypes. Therefore, the IC-RT-LAMP assay can serve as a useful tool in the management of potentially severe strains of CTV.

Development of a colorimetric RT-LAMP amplification assay adapted to an early and easy detection of Dengue virus

Background: Dengue viruses (DENV) produce one of the most important emerging viral diseases around the world. This mosquito-borne disease has the greater impact in tropical/subtropical areas, causing symptoms ranging from acute, self-limiting febrile illness, dengue fever, to severe dengue hemorrhagic fever to dengue shock syndrome. The infection is caused by 4 distinct, but closely related, serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). There are neither specific antiviral drugs nor approved vaccines; then, an early detection is a crucial step for proper patient management, lowing fatality rates, and for preventing the spread of the disease. The aim of this work was to develop a simplified test for DENV detection in early stages (during the febrile period of the infection), identifying the serotype involved. Methods & Materials: We developed and evaluated a colorimetric reverse transcriptase loop-mediated isothermal amplification assay (ColorRT- LAMP), an alternative method of polymerase chain reaction (PCR) that, as it works at a fixed temperature, not require thermocyclers and, also, can be performed in short times, showing accurate and reliable results. The complete reaction was performed in one step in a single tube by mixing primers, reverse-transcriptase and DNA polymerase together with the tested samples (RNA genomes from the international references of Dengue serotypes). Ranges of temperature and time were evaluated, selecting 65 ∘C for 60 min as the best reaction options. The read out of the test was defined as a color change -visible to a naked eye-, by the addition of a visible pH indicator dye, the neutral red dye, prior to the amplification; this pH indicator changes its color as a result of the amplification performed under minimal buffering conditions. Results: Here we described the achievement of an early and easy DENV detection test. The results showed that our ColorRT- LAMP test is highly sensitive, more than quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and highly specific, differentiating between the four serotypes and not showing cross reaction with another closely related arbovirus (Zika). Conclusion: Here we described the development of a simple ColorRT- LAMP test that could be used as a powerful tool for an early, easy, and sensible detection of Dengue disease.

Use of reverse transcriptase loop-mediated isothermal amplification assay for field detection of Newcastle disease virus using less invasive samples

A novel nucleic acid amplification method, loop-mediated isothermal amplification, was developed and recently demonstrated detection of Newcastle disease virus (NDV) in tissue samples. But slaughter of poultry for test samples is often faced with resentment by low-income farmers. This study was undertaken to determine the test properties of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) in detection of NDV in clinical cases using cloacal and oropharyngeal swabs. Samples included 46 tracheal tissues, 94 cloacal and 107 oro-pharyngeal swabs from on-station and 30 spleens, 74 cloacal and 74 oro-pharyngeal swabs from the field. Analysis was done using specific RT-LAMP targeting the fusion (F) protein. While the method detected NDV from swab samples, no RNA of other poultry disease viruses was amplified, indicating analytical specificity of 100%. RT-LAMP took ≤36 minutes in 83% (n=329) of positive reactions with all samples amplified in

Optimisation of reverse transcriptase loop-mediated isothermal amplification assay for rapid detection of Macrobrachium rosenbergii noda virus and extra small virus in Macrobrachium rosenbergii

The standardisation and optimisation of a one step single tube reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) procedure is described for rapid diagnosis of white tail disease, a viral disease caused by Macrobrachium rosenbergii noda virus ( MrNV) and extra small virus (XSV), in giant fresh water prawn, M. rosenbergii. Time, temperature and quantity of each reagent were optimised for the detection of the two viruses. This method was more sensitive than the conventional reverse transcriptase polymerase chain reaction (RT-PCR) for detecting the two viruses. The RT-LAMP reaction is highly suited for disease diagnosis in developing countries. Amplification of DNA can be detected without the use of agarose gel electrophoresis, by the production of a whitish precipitate of magnesium pyrophosphate as a by-product. The cost of RT-LAMP for one reaction is nearly 4 times less than that of RT-PCR.

A one-step reverse transcriptase loop-mediated isothermal amplification assay for simple and rapid detection of swine vesicular disease virus

This report describes the development of a one-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for the detection of swine vesicular disease virus (SVDV). The assay detects the virus rapidly, within 30–60 min and the result is visualised either by gel-electrophoresis or by the naked eye through the addition of SybrGreen. A collection of 28 SVDV isolates were tested positive, while heterologous viruses such as foot-and-mouth disease virus and vesicular stomatitis virus remained negative. The performance of the RT-LAMP was compared directly with real-time PCR using RNA from clinical samples including nasal swabs, serum and faeces. For nasal swabs and serum the sensitivity of the RT-LAMP was shown to be at least equivalent to real-time PCR. Interestingly, for faecal samples the RT-LAMP assay was shown to be even more sensitive than real-time PCR, possibly because it is less sensitive to inhibitory substances. This RT-LAMP assay provides a number of benefits for the diagnosis of SVD, since the assay is sensitive and rapid, and the isothermal amplification strategy used is not reliant upon expensive equipment it is particularly suited for “front line” diagnosis of SVD in modestly equipped laboratories, in field stations or in mobile diagnostic units.