End-point rapid detection of total and pathogenic Vibrio parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) in raw seafood using a colorimetric loop-mediated isothermal amplification-xylenol orange technique.

Background: Vibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis in humans worldwide. To ensure seafood safety and to minimize the occurrence of seafood-borne diseases, early detection of total V. parahaemolyticus (pathogenic and non-pathogenic strains) and pathogenic V. parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) is required. This study further improved a loop-mediated isothermal amplification (LAMP) assay using xylenol orange (XO), a pH sensitive dye, to transform conventional LAMP into a one-step colorimetric assay giving visible results to the naked eye. LAMP-XO targeted rpoD for species specificity and tdh, trh1, and trh2 for pathogenic strains. Multiple hybrid inner primers (MHP) of LAMP primers for rpoD detection to complement the main primer set previously reported were designed by our group to maximize sensitivity and speed. Methods: Following the standard LAMP protocol, LAMP reaction temperature for rpoD, tdh, trh1, and trh2 detection was first determined using a turbidimeter. The acquired optimal temperature was subjected to optimize six parameters including dNTP mix, betaine, MgSO4, Bst 2.0 WarmStart DNA polymerase, reaction time and XO dye. The last parameter was done using a heat block. The color change of the LAMP-XO result from purple (negative) to yellow (positive) was monitored visually. The detection limits (DLs) of LAMP-XO using a 10-fold serial dilution of gDNA and spiked seafood samples were determined and compared with standard LAMP, PCR, and quantitative PCR (qPCR) assays. Subsequently, the LAMP-XO assay was validated with 102 raw seafood samples and the results were compared with PCR and qPCR assays. Results: Under optimal conditions (65 °C for 75 min), rpoD-LAMP-XO and tdh-LAMP-XO showed detection sensitivity at 102 copies of gDNA/reaction, or 10 folds greater than trh1-LAMP-XO and trh2-LAMP-XO. This level of sensitivity was similar to that of standard LAMP, comparable to that of the gold standard qPCR, and 10-100 times higher than that of PCR. In spiked samples, rpoD-LAMP-XO, tdh-LAMP-XO, and trh2-LAMP-XO could detect V. parahaemolyticus at 1 CFU/2.5 g spiked shrimp. Of 102 seafood samples, LAMP-XO was significantly more sensitive than PCR (P < 0.05) for tdh and trh2 detection and not significantly different from qPCR for all genes determined. The reliability of tdh-LAMP-XO and trh2-LAMP-XO to detect pathogenic V. parahaemolyticus was at 94.4% and 100%, respectively. Conclusions: To detect total and pathogenic V. parahaemolyticus, at least rpoD-LAMP-XO and trh2-LAMP-XO should be used, as both showed 100% sensitivity, specificity, and accuracy. With short turnaround time, ease, and reliability, LAMP-XO serves as a better alternative to PCR and qPCR for routine detection of V. parahaemolyticus in seafood. The concept of using a one-step LAMP-XO and MHP-LAMP to enhance efficiency of diagnostic performance of LAMP-based assays can be generally applied for detecting any gene of interest.

Enhancement of loop mediated isothermal amplification's sensitivity and speed by multiple inner primers for more efficient identification of Vibrio parahaemolyticus.

The modified loop-mediated isothermal amplification (LAMP), called multiple hybrid, inner primers (MHP)-LAMP, was developed to enhance the efficiency of the existing LAMP-based assay for Vibrio parahaemolyticus detection. The method was built on a conventional LAMP assay by employing 2 newly designed extra sets of primers to increase the initial binding sites of core primers on the V. parahaemolyticus's rpoD gene from 8 to 12. With this strategy, the assay detection sensitivity was increased by 10 folds, with the detection limit (DL) approaching 100 copies of purified target genomic DNA (gDNA) as analyzed by real-time turbidity measurement and gel electrophoresis. The MHP also accelerated the rate of DNA amplification by 30%, rendering the assay faster. The MHP-LAMP assay did not cross- react with other pathogens, indicating that it was highly specific for V. parahaemolyticus detection. Whilst V. parahaemolyticus was used as a study model herein, our idea of using MHP to maximize assay sensitivity and speed is considered as a universal strategy that can be applied to enhance efficiency of LAMP-based assays for detecting any DNA and RNA of interest. •The strategy of using multiple hybrid, inner primers (MHP) to enhance LAMP assay's efficiency was demonstrated with success.•The MHP enhanced the sensitivity and speed of the existing LAMP assay, designed to detect V. parahaemolyticus, by 10 times and 30%, respectively.•The proposed strategy can be applied to boost up any other LAMP-based assay's diagnostic performance.

One-step colorimetric isothermal detection of COVID-19 with AI-assisted automated result analysis: A platform model for future emerging point-of-care RNA/DNA disease diagnosis.

Colorimetric loop-mediated DNA isothermal amplification-based assays have gained momentum in the diagnosis of COVID-19 owing to their unmatched feasibility in low-resource settings. However, the vast majority of them are restricted to proprietary pH-sensitive dyes that limit downstream assay optimization or hinder efficient result interpretation. To address this problem, we developed a novel dual colorimetric RT-LAMP assay using in-house pH-dependent indicators to maximize the visual detection and assay simplicity, and further integrated it with the artificial intelligence (AI) operated tool (RT-LAMP-DETR) to enable a more precise and rapid result analysis in large scale testing. The dual assay leverages xylenol orange (XO) and a newly formulated lavender green (LG) dye for distinctive colorimetric readouts, which enhance the test accuracy when performed and analyzed simultaneously. Our RT-LAMP assay has a detection limit of 50 viral copies/reaction with the cycle threshold (Ct) value ≤ 39.7 ± 0.4 determined by the WHO-approved RT-qPCR assay. RT-LAMP-DETR exhibited a complete concordance with the results from naked-eye observation and RT-qPCR, achieving 100% sensitivity, specificity, and accuracy that altogether render it suitable for ultrasensitive point-of-care COVID-19 screening efforts. From the perspective of pandemic preparedness, our method offers a simpler, faster, and cheaper (∼$8/test) approach for COVID-19 testing and other emerging pathogens with respect to RT-qPCR.

Establishing a gold standard method for the detection of Cherax reovirus using reverse transcriptase, quantitative, polymerase chain reaction.

Cherax reovirus infects redclaw crayfish (Cherax quadricarinatus) and it may be involved in mortalities between 5-20 % and stunting of up to 40 % of survivors. The sequence of the RNA-dependent RNA polymerase was used to develop a reverse transcription, quantitative, PCR (RT-qPCR) which was specific against seven other crustacean viruses (Athtab bunyavirus, Chequa iflavirus, Macrobrachium rosenbergii nodavirus, Gill-associated virus, Taura syndrome virus, White spot syndrome virus, and Penaeus stylirostris Penstylhamaparvovirus) although GAV produced a reaction that was easily separated by melt curve analysis. A strong linear correlation (r2 = 0.9965) was obtained between viral quantities ranging from 107 to 10 viral copies/reaction with an amplification efficiency of 0.92. This RT-qPCR is 2-times faster and 100 times more sensitive than a standard RT-PCR using agarose gel electrophoresis with the potential to detect the virus down to 7.64 copies/reaction in clinical samples. In clinical crayfish samples, it was able to detect Cherax reovirus in crayfish when the traditional RT-PCR was negative. Its' measurement of uncertainty was less than 2% (0.02-1.9), similar to PCRs for other crustacean viruses. This RT-qPCR is proposed as the gold standard and should be used for the screening of populations of C. quadricarinatus for broodstock before being used in hatcheries or on farms.

Ultrasensitive detection of Mycobacterium tuberculosis by a rapid and specific probe-triggered one-step, simultaneous DNA hybridization and isothermal amplification combined with a lateral flow dipstick.

Mycobacterium tuberculosis (Mtb) is an insidious scourge that has afflicted millions of people worldwide. Although there are many rapid methods to detect it based on loop-mediated isothermal amplification (LAMP) and a lateral flow dipstick (LFD), this study made further improvements using a new set of primers to enhance LAMP performance and a novel DNA probe system to simplify detection and increase specificity. The new probe system eliminates the post-LAMP hybridization step typically required for LFD assays by allowing co-hybridization and amplification of target DNA in one reaction while preventing self-polymerization that could lead to false-positive results. The improved assay was named Probe-Triggered, One-Step, Simultaneous DNA Hybridization and LAMP Integrated with LFD (SH-LAMP-LFD). SH-LAMP-LFD was simpler to perform and more sensitive than previously reported LAMP-LFD and PCR methods by 100 and 1000 times, respectively. It could detect a single cell of Mtb. The absence of cross-reactivity with 23 non-TB bacteria, and accurate test results with all 104 blind clinical samples have highlighted its accuracy. Its robustness and portability make SH-LAMP-LFD suitable for users in both low and high resource settings.

Graphene-based electrochemical genosensor incorporated loop-mediated isothermal amplification for rapid on-site detection of Mycobacterium tuberculosis.

Tuberculosis (TB) is one of the most contagious and lethal infectious diseases that affects more than 10 million individuals worldwide. A lack of rapid TB diagnosis is partly responsible for its alarming spread and prevalence in many regions. To address this problem, we report a novel integrated point-of-care platform to detect a TB-causative bacterium, Mycobacterium tuberculosis (Mtb). This leverages loop-mediated isothermal amplification (LAMP) for Mtb-DNA amplification and the screen-printed graphene electrode (SPGE) for label-free electrochemical analysis of DNA amplicons. When implemented on a portable potentiostat device developed in-house, the system (LAMP-EC) offers a rapid end-point qualitative analysis of specific DNA amplicons that will be displayed as a discrete positive/negative readout on the LCD screen. Under optimized conditions, LAMP-EC showed a comparable detection limit to the previously developed LAMP assay with a lateral flow readout at 1 pg total DNA or 40 Mtb genome equivalents. This highly specific technique detected the presence of TB in all 104 blinded sputum samples with a 100% accuracy. Our technique can also easily be clinically adopted due to its affordability (∼USD2.5/test), rapidity (<65 min turnaround time) and feasibility (lack of advanced instrumental requirement). This serves as a practical incentive, appealing to users in both high- and low-resource settings across the TB endemic regions and economic backgrounds.

Xylenol orange-based loop-mediated DNA isothermal amplification for sensitive naked-eye detection of Escherichia coli.

Loop-mediated isothermal amplification (LAMP) can amplify DNA specifically and sensitively. Under minimal buffering conditions, it produces hydrogen ions that lower the pH of the solution upon DNA amplification. This characteristic was applied to visually detect amplified DNA of Escherichia coli through the use of Xylenol Orange, a pH-dependent dye. Under the optimal conditions, 120 min at 63 °C, the Xylenol orange-dependent colorimetric LAMP revealed a detection limit as low as 1 CFU, namely 100,000 times more sensitive than typical multiplex PCR, and showed no cross-reactions with other foodborne pathogens. The colorimetric assay was successfully exploited to detect E. coli contaminations in milk samples, showing high reliability and the same high sensitivity with naked-eye readout. Together with robustness, simplicity, and visual detectability of amplification, this assay can serve as an alternative tool to PCR for detecting E. coli, which is suitable for both laboratory and on-field applications.

Recombinase polymerase amplification combined with a lateral flow dipstick for discriminating between infectious Penaeus stylirostris densovirus and virus-related sequences in shrimp genome.

Penaeus stylirostris densovirus (PstDV) is an important shrimp pathogen that causes mortality in P. stylirostris and runt deformity syndrome (RDS) in Penaeus vannamei and Penaeus monodon. Recently, PstDV-related sequences were found in the genome of P. monodon and P. vannamei. This led to false positive results by PCR-based detection system. Here, a more efficient detection platform based on recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) was developed for detecting PstDV. Under the optimal conditions, 30 min at 37°C for RPA followed by 5 min at room temperature for LFD, the protocol was 10 times more sensitive than the Saksmerphrome et al's interim 3-tube nested PCR and showed no cross-reaction with other shrimp viruses. It also reduced false positive results arising from viral inserts to ∼5% compared to 76-78% by the IQ2000™ nested PCR kit and the 309F/R PCR protocol currently recommended by World Organization for Animal Health (OIE) for PstDV detection. Together with simplicity and portability, the protocol serves as an alternative tool to PCR for primarily screening PstDV, which is suitable for both laboratory and field application.

Separation of endogenous viral elements from infectious Penaeus stylirostris densovirus using recombinase polymerase amplification.

Non-infectious Penaeus stylirostris densovirus (PstDV)-related sequences in the shrimp genome cause false positive results with current PCR protocols. Here, we examined and mapped PstDV insertion profile in the genome of Australian Penaeus monodon. A DNA sequence which is likely to represent infectious PstDV was also identified and used as a target sequence for recombinase polymerase amplification (RPA)-based approach, developed for specifically detecting PstDV. The RPA protocol at 37 °C for 30 min showed no cross-reaction with other shrimp viruses, and was 10 times more sensitive than the 309F/R PCR protocol currently recommended by the World Organization for Animal Health (OIE) for PstDV diagnosis. These features, together with the simplicity of the protocol, requiring only a heating block for the reaction, offer opportunities for rapid and efficient detection of PstDV.

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.

Rapid and sensitive detection of shrimp yellow head virus by loop-mediated isothermal amplification combined with a lateral flow dipstick.

Yellow head virus (YHV) is a highly virulent pathogen that has caused severe mortality in cultivated shrimp (Penaeus monodon and Penaeus vannamei) in Thailand. There are several technologies that are applied to detect YHV for further control of the disease. RT-PCR is currently widely used in the laboratory, but it has some disadvantages related to cost, time-consuming and complexity. An alternative assay combines RT with loop-mediated isothermal amplification (LAMP) that not only provides high specificity, sensitivity and rapidity, but is also cheaper and more suitable for field applications in shrimp aquaculture than the RT-PCR. RT-LAMP is performed under isothermal conditions with a set of four to six primers designed to recognize six to eight distinct target sequences, and it has been combined with a chromatographic lateral-flow dipstick (LFD) to detect LAMP amplified product, which avoids the use of gel electrophoresis. In this study, RT-LAMP for the detection of YHV was developed by isothermal amplification at 65 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min (time required approximately 55 min, excluding RNA extraction and preparation time). The detection limit of RT-LAMP-LFD was 0.1 pg RNA extracted from shrimp infected with YHV equivalent to the nested RT-PCR, and no cross reaction was observed with other common shrimp viral pathogens. The LAMP method described in this study showed a rapid, high sensitivity and specificity and it is recommended as user-friendly for diagnosis of YHV in the field.

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 shrimp yellow head virus using loop-mediated isothermal amplification and a colorogenic nanogold hybridization probe.

Salt-induced self-aggregation of gold nanoparticles (AuNP) carrying unisense ssDNA probes can be prevented specifically by complementary DNA. Loop-mediated isothermal amplification (LAMP) can amplify DNA rapidly. Here, the two techniques were combined to detect yellow head virus (YHV). The LAMP-AuNP method required 60 min for LAMP and 5 min for hybridization of LAMP products to an AuNP-labeled ssDNA probe followed by salt induced probe-particle aggregation to visualize color development. The detection sensitivity of the method was comparable to that of the commercial IQ2000™ nested RT-PCR but only required ~65 min to produce a result, and did not cross-detect other shrimp viruses. As the LAMP-AuNP protocol only requires a heating block, it offers opportunities for rapid detection of YHV.

Detection of shrimp Taura syndrome virus by loop-mediated isothermal amplification using a designed portable multi-channel turbidimeter.

In this study, a portable turbidimetric end-point detection method was devised and tested for the detection of Taura syndrome virus (TSV) using spectroscopic measurement of a loop-mediated isothermal amplification (LAMP) by-product: magnesium pyrophosphate (Mg(2)P(2)O(7)). The device incorporated a heating block that maintained an optimal temperature of 63°C for the duration of the RT-LAMP reaction. Turbidity of the RT-LAMP by-product was measured when light from a light-emitting diode (LED) passed through the tube to reach a light dependent resistance (LDR) detector. Results revealed that turbidity measurement of the RT-LAMP reactions using this device provided the same detection sensitivity as the agarose gel electrophoresis detection of RT-LAMP and nested RT-PCR (IQ2000™) products. Cross reactions with other shrimp viruses were not found, indicating that the RT-LAMP-turbidity measurement was highly specific to TSV. The combination of 10 min for rapid RNA preparation with 30 min for RT-LAMP amplification followed by turbidity measurement resulted in a total assay time of less than 1h compared to 4-8h for the nested RT-PCR method. RT-LAMP plus turbidity measurement constitutes a platform for the development of more rapid and user-friendly detection of TSV in the field.

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.

Experimental infections reveal that common Thai crustaceans are potential carriers for spread of exotic Taura syndrome virus.

Taura syndrome virus (TSV) was first reported as a serious cause of shrimp mortality limited to reared Penaeus (Litopenaeus) vannamei in the Americas, where it spread principally through regional and international transfer of live post larvae (PL) and broodstock. Subsequently, through importation of infected broodstock, TSV outbreaks spread to Asia, first to Taiwan and China and then to Thailand, Indonesia and Korea. Since its introduction to Thailand, outbreaks have occasionally been reported from rearing ponds stocked with batches of specific pathogen free (SPF) P. vannamei PL that tested negative for TSV by nested RT-PCR assay. Since it was possible that the outbreaks may have occurred via horizontal transfer of TSV from wild carrier species, we tested 5 common native crustaceans that live in and around shrimp ponds (2 palaemonid shrimp species, Palaemon styliferus and Macrobrachium lanchesteri, and 3 species of crabs, Sesarma mederi, Scylla serrata and Uca vocans) for susceptibility to TSV in experimental challenges. We found that U. vocans, S. serrata and S. mederi did not die but, respectively, gave strong RT-PCR reactions indicating heavy viral load at 5, 10 and 15 d post-injection of TSV and 10, 15 and up to 50 d after feeding with TSV-infected P. vannamei carcasses. Also after feeding, P. styliferus did not die, but a high proportion gave strong RT-PCR reactions at 5 d post-challenge and no reactions at 15 d. Similarly after feeding, M. lanchesteri showed no mortality and gave only light RT-PCR reactions at 2 d, moderate reactions at 5 d and no reaction at 15 d. By contrast, transmission experiments from the TSV-infected crabs and palaemonid shrimp via water or feeding resulted in death of all the exposed P. vannamei from 8 to 12 d post-challenge and all were positive for heavy viral load by RT-PCR assay. Despite the results of these laboratory challenge tests, natural TSV infections were not detected by nested RT-PCR in samples of these species taken from the wild. These results indicated that transmission of TSV from infected crabs and palaemonid shrimp via water or feeding might pose a potential risk to shrimp aquaculture.

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.