LAMP Primers Research Articles

SARS-CoV-2 Identification Using Colorimetric Loop-Mediated Isothermal Amplification

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused worldwide losses from all points of view. Although there are efficient vaccines against this virus, rapid detection remains essential for controlling its spread. The gold standard for the COVID-19 diagnostic is the use of quantitative real time polymerase chain reaction (RT-qPCR) from naso-/oro-pharyngeal swabs and, alternatively, saliva specimens. A faster alternative to the RT-qPCR method, the colorimetric reverse transcription loop-mediated isothermal amplification (cLAMP) has a good sensitivity and specificity and has been proposed as an efficient screening method. In this study, we analyzed several sets of LAMP primers for COVID-19 testing using purified RNA samples and raw saliva samples, and tested several reaction conditions. Our results showed that the sensitivity of cLAMP reactions on raw saliva samples can be significantly augmented by the addition of guanidine hydrochloride to levels, which allow its use for COVID-19 screening.

Mpl-Gene-Based Loop-Mediated Isothermal Amplification Assay for Specific and Rapid Detection of Listeria monocytogenes in Various Food Samples.

Listeria monocytogenes represents a high risk in food and can trigger potentially fatal listeriosis. The objective of this study was to detect L. monocytogenes in food using the LAMP method in a fast, specific, sensitive manner and thus to preventively test food for the presence of the target species. The reaction was performed and established using the portable real-time fluorometer Genie® II (OptiGene Ltd., Horsham, United Kingdom). In this new assay, six LAMP primers targeted the mpl-gene sequence of L. monocytogenes. A total of 148 different isolates, including 105 L. monocytogenes and 43 non-L. monocytogenes strains, were tested. Analytical sensitivity was determined based on different DNA- and cell concentrations. The detection limit with a detection rate of 100% was 5 pg of DNA or 275 colony-forming units (CFU) per reaction. Artificially contaminated minced beef and grated mozzarella were also tested. The assay was 100% successful to detect an initial bacterial contamination of 0.4-4 CFU g-1 food after 24 h enrichment in half-Fraser broth. Finally, natively contaminated samples were tested in comparison to the microbiological reference method and real-time polymerase chain reaction. Native sample testing revealed 100% consistent findings between LAMP and the standard culture method after first enrichment for 24 h. In addition, a rapid colony-confirmation method was established that enabled reliable identification of L. monocytogenes isolates on different selective culture media using a simplified DNA extraction by boiling. This study showed that the developed assay was able to determine whether a food is safe with respect to the food-safety criteria of 100 CFU per gram, according to standards of the European Union, for L. monocytogenes and provided faster results than the cultural reference method.

Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis.

Isothermal techniques with lateral flow detection have emerged as a point of care (POC) technique for malaria, a major parasitic disease in tropical countries such as India. Plasmodium falciparum and Plasmodium vivax are the two most prevalent malaria species found in the country. An advanced multiplex loop-mediated isothermal amplification (mLAMP) combined with a lateral flow dipstick (LFD) technique was developed for the swift and accurate detection of P. falciparum and P. vivax, overcoming the challenges of the existing RDTs (rapid diagnostic tests). A single set of LAMP primers with a biotinylated backward inner primer (BIP primer) was used for DNA amplification of both malaria species in a single tube. The amplified DNA was hybridized with fluorescein isothiocyanate (FITC) and digoxigenin-labelled DNA probes, having a complemented sequence for the P. falciparum and P. vivax genomes, respectively. A colour band appeared on two separate LFDs for P. falciparum and P. vivax upon running the hybridized solution over them. In total, 39 clinical samples were collected from ICMR-NIMR, New Delhi. Melting curve analysis, with cross primers for both species, was used to ascertain specificity, and the sensitivity was equated with a polymerase chain reaction (PCR). The results were visualized on the LFD for both species within 60 min. We found 100% sensitivity and specificity, when compared with a traditional PCR. Melting curve analysis of mLAMP revealed the lowest detection limit of 0.15 pg/μL from sample genomic DNA. The mLAMP-LFD assays could be a potential point of care (POC) tool for early diagnosis in non-laboratory conditions, with the convenience of a reduced assay time and the simple interpretation of results.

Developing RT-LAMP assays for rapid diagnosis of SARS-CoV-2 in saliva.

SummaryBackgroundThe coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has killed millions of people worldwide. The current crisis has created an unprecedented demand for rapid test of SARS-CoV-2 infection.MethodsReverse transcription loop-mediated isothermal amplification (RT-LAMP) is a fast and convenient method to amplify and identify the transcripts of a targeted pathogen. However, the sensitivity and specificity of RT-LAMP were generally regarded as inferior when compared with the gold standard RT-qPCR. To address this issue, we combined bioinformatic and experimental analyses to improve the assay performance for COVID-19 diagnosis.FindingsFirst, by experimental screening as well as high-throughput sequencing studies, we discovered new primer features that impacted LAMP sensitivity and specificity. These features were then used to build an improved bioinformatics algorithm to design LAMP primers targeting SARS-CoV-2. We further rigorously validated these new assays for their efficacy and specificity. We demonstrated that multiplexed RT-LAMP assay could directly detect as low as 1.5 copies/µL of SARS-CoV-2 particles in saliva, without the need of RNA isolation. We further tested this ultra-sensitive and specific RT-LAMP assay using saliva samples from COVID-19 patients. Clinical validation results indicated that the new RT-LAMP assay was comparable to standard RT-qPCR in overall assay sensitivity and specificity.InterpretationIn summary, our new LAMP primer design algorithm along with the validated assays provide a fast and reliable method for the diagnosis of COVID-19 cases.FundingNational Institutes of Health.

Detection of MGMT methylation status using a Lab-on-Chip compatible isothermal amplification method.

The growing cancer burden necessitates the development of cost-effective solutions that provide rapid, precise and personalised information to improve patient outcome. The aim of this study was to develop a novel, Lab-on-Chip compatible method for the detection and quantification of DNA methylation for MGMT, a well-established molecular biomarker for glioblastoma, with direct clinical translation as a predictive target. A Lab-on-Chip compatible isothermal amplification method (LAMP) was used to test its efficacy for detection of sequence-specific methylated regions of MGMT, with the method's specificity and sensitivity to have been compared against gold-standards (MethyLight, JumpStart). Our LAMP primer combinations were shown to be specific to the MGMT methylated region, while sensitivity assays determined that the amplification methods were capable of running at clinically relevant DNA concentrations of 0.2 - 20 ng/µL. For the first time, the ability to detect the presence of DNA methylation on bisulfite converted DNA was demonstrated on a Lab-on-Chip setup, laying the foundation for future applications of this platform to other epigenetic biomarkers in a point-of-care setting.

LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time fluorescence or lateral flow test.

ACRISPR-based nucleic acid detection platform, termed LACD (loop-mediated isothermal amplification coupled with CRISPR-Cas12a-mediated diagnostic)has been developed. In the LACD system, the core primer used in conventional LAMP (forward inner primer or backward inner primer) was engineered to contain a PAM (protospacer adjacent motif) site (TTTT) at the linker region. As a result, the LAMP amplicons contained a specific PAM site for CRISPR-Cas12a recognition. At the CRISPR-mediated detection stage, the resulting LAMP products can activate the corresponding CRISPR-Cas12a effector upon the formation of the CRISPR-Cas12a/gRNA/target DNA complex. The single-strand DNA (ssDNA) reporter molecules are then rapidly cleaved due to the CRISPR-Cas12a's trans-enzyme activity. The ssDNA degradation can then be visualized on a lateral flow biosensor or measured by a real-time fluorescence instrument. Our LACD assay allows any target sequence to be detected (even targets which do not contain any PAM sites) as long as they met the design requirement for LAMP. The feasibility of the LACD methodology for nucleic acid detection was validated on the Mycobacterium tuberculosis complex (MTC). This proof-of-concept assay can be reconfigured to detect a variety of target sequences by redesigning the engineered LAMP primers.

Nanoparticle-Based Lateral Flow Biosensors Integrated With Loop-Mediated Isothermal Amplification for the Rapid and Visual Diagnosis of Hepatitis B Virus in Clinical Application.

Hepatitis B virus (HBV) infection remains one of the major public health issues worldwide. Developing a rapid, sensitive, specific, easy-to-operate, and cost-saving approach for the diagnosis of HBV is essential for its therapy and prevention. Here, we first devised a novel approach, termed “loop-mediated isothermal amplification integrated with a nanoparticle-based lateral flow biosensor (LAMP-LFB),” for the detection of HBV in clinical application. The results indicated that a set of LAMP primers based on the S gene were valid for the establishment of HBV-LAMP-LFB. The optimal HBV-LAMP can be carried out at a constant temperature of 65°C for 40 min. The whole detection process, including HBV genomic DNA preparation (∼10 min), LAMP (40 min), and LFB reading (within 2 min), can be accomplished within 60 min. The limit of detection of the HBV-LAMP-LFB assay was 7.5 IU per test. The specificity of this assay was one hundred percent, and there was no cross-reactivity with other pathogens. Hence, these results indicated that the HBV-LAMP-LFB assay established in the current study is a sensitive, rapid, specific, visual, simple, and cost-saving method for the screening of HBV agents. More importantly, the HBV-LAMP-LFB has remarkable potential to develop a point-of-care testing in clinical application, especially in resource-scarce regions.

Optimization and validation of RT-LAMP assay for diagnosis of SARS-CoV2 including the globally dominant Delta variant

BackgroundSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 pandemic, has infected more than 179 million people worldwide. Testing of infected individuals is crucial for identification and isolation, thereby preventing further spread of the disease. Presently, Taqman™ Reverse Transcription Real Time PCR is considered gold standard, and is the most common technique used for molecular testing of COVID-19, though it requires sophisticated equipments, expertise and is also relatively expensive.ObjectiveDevelopment and optimization of an alternate molecular testing method for the diagnosis of COVID-19, through a two step Reverse Transcription Loop-mediated isothermal AMPlification (RT-LAMP).ResultsPrimers for LAMP were carefully designed for discrimination from other closely related human pathogenic coronaviruses. Care was also taken that primer binding sites are present in conserved regions of SARS-CoV2. Our analysis shows that the primer binding sites are well conserved in all the variants of concern (VOC) and variants of interest (VOI), notified by World Health Organization (WHO). These lineages include B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427/B.1.429, P.2, B.1.525, P.3, B.1.526 and B.1.617.1. Various DNA polymerases with strand displacement activity were evaluated and conditions were optimized for LAMP amplification and visualization. Different LAMP primer sets were also evaluated using synthetic templates as well as patient samples.ConclusionIn a double blind study, the RT-LAMP assay was validated on more than 150 patient samples at two different sites. The RT-LAMP assay appeared to be 89.2% accurate when compared to the Taqman™ rt-RT-PCR assay.

Visual and Rapid Diagnosis of Neisseria gonorrhoeae Using Loop-Mediated Isothermal Amplification Combined With a Polymer Nanoparticle-Based Biosensor in Clinical Application.

Neisseria gonorrhoeae is a host-adapted human pathogen that causes sexually transmitted gonorrhea and remains to be a serious global public health challenge, especially in low- and middle-income regions. It is vital to devise a reliable, simple, cost-saving, and easy-to-use assay for detecting the N. gonorrhoeae agent. In the current study, we firstly report a novel approach, loop-mediated isothermal amplification linked with a polymer nanoparticle–based biosensor (LAMP-PNB), that was used for identifying N. gonorrhoeae in clinical samples. The results showed that the LAMP primers based on the orf1 gene were valid for development of the N. gonorrhoeae-LAMP-PNB assay. The detection system with optimal conditions could be performed at a fixed temperature of 64°C for 40 min. The whole process, including genomic DNA preparation (approximately 10 min), LAMP reaction (40 min), and PNB reporting (approximately 2 min), could be accomplished within 60 min. The limit of detection (LoD) of the N. gonorrhoeae-LAMP-PNB assay was 50 copies per test. The specificity of the current assay was 100%, and no cross-reactions to non–N. gonorrhoeae isolates were observed. These results confirmed that the N. gonorrhoeae-LAMP-PNB technique is a reliable, specific, sensitive, rapid, low-cost, and easy-to-use method for detecting gonococci isolates. More importantly, this assay has great potential to develop a point-of-care (POC) testing method in clinical practice, especially in resource-constrained regions.

A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major threat to public health. Rapid molecular testing for convenient and timely diagnosis of SARS-CoV-2 infections represents a challenge that could help to control the current pandemic and prevent future outbreaks. We aimed to develop and validate a multiplex and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using lyophilized LAMP reagents for sensitive and rapid detection of SARS-CoV-2. LAMP primers were designed for a set of gene targets identified by a genome-wide comparison of viruses. Primer sets that showed optimal features were combined into a multiplex RT-LAMP assay. Analytical validation included assessment of the limit of detection (LoD), intra- and inter-assay precision, and cross-reaction with other respiratory pathogens. Clinical performance compared to that of real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was assessed using 278 clinical RNA samples isolated from swabs collected from individuals tested for COVID-19. The RT-LAMP assay targeting the RNA-dependent RNA polymerase (RdRp), membrane (M), and ORF1ab genes achieved a comparable LoD (0.65 PFU/mL, CT=34.12) to RT-qPCR and was 10-fold more sensitive than RT-qPCR at detecting viral RNA in clinical samples. Cross-reactivity to other respiratory pathogens was not observed. The multiplex RT-LAMP assay demonstrated a strong robustness and acceptable intra- and inter-assay precision (mean coefficient of variation, 4.75% and 8.30%). Diagnostic sensitivity and specificity values were 100.0% (95% CI: 97.4–100.0%) and 98.6% (95% CI: 94.9–99.8%), respectively, showing high consistency (Cohen’s kappa, 0.986; 95% CI: 0.966–1.000; p<0.0001) compared to RT-qPCR. The novel one-step multiplex RT-LAMP assay is storable at room temperature and showed similar diagnostic accuracy to conventional RT-qPCR, while being faster (<45 min), simpler, and cheaper. The new assay could allow reliable and early diagnosis of SARS-CoV-2 infections in primary health care. It may aid large-scale testing in resource-limited settings, especially if it is integrated into a point-of-care diagnostic device.

Highly specific and sensitive detection of the Mycobacterium tuberculosis complex using multiplex loop-mediated isothermal amplification combined with a nanoparticle-based lateral flow biosensor.

Tuberculosis (TB) is the deadliest infectious caused by Mycobacterium tuberculosis complex (MTBC). Because most TB cases occur within low-income populations, developing a specific, sensitive, cost-saving, and rapid point-of-care test for the early diagnosis of TB is important for achieving the WHO's End Tuberculosis Strategy. In the current study, a novel nucleic acid detection strategy that includes multiplex loop-mediated isothermal amplification combined with a nanoparticle-based lateral flow biosensor (mLAMP-LFB) was used to detect MTBC. The two sets of LAMP primers specific to the IS6110 and gyrB genes of MTBC were successfully designed and validated for the detection of MTBC. The preferred reaction conditions for this assay were confirmed to be 65°C for 40min, and the amplification products could be visually identified through LFB within 2min. The full assay process, including genomic DNA template extraction, LAMP reaction, and product detection, could be completed in 80min. The limit detection of the assay was 100fg of DNA in pure culture. The specificity of the assay was 100%, and it had no cross-reactions to other strains. Thus, the m-LAMP-LFB technology established in the present study was an objective, rapid, simple, and sensitive assay for MTBC identification, which could be applied in a clinical setting, especially in resource-constrained regions of the world.

Evaluation of different DNA extraction methods and loop-mediated isothermal amplification primers for the detection of Mycobacterium ulcerans in clinical specimens

BackgroundEarly diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories.MethodsIn this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms.ResultsOur results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75–91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings.ConclusionsFor further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50–91.49%) and specificity (89.23–100%), depending on the DNA extraction methods used.

Multicopy targets for Plasmodium vivax and Plasmodium falciparum detection by colorimetric LAMP

BackgroundLoop-mediated isothermal amplification (LAMP) for malaria diagnosis at the point of care (POC) depends on the detection capacity of synthesized nucleic acids and the specificity of the amplification target. To improve malaria diagnosis, new colorimetric LAMP tests were developed using multicopy targets for Plasmodium vivax and Plasmodium falciparum detection.MethodsThe cytochrome oxidase I (COX1) mitochondrial gene and the non-coding sequence Pvr47 for P. vivax, and the sub-telomeric sequence of erythrocyte membrane protein 1 (EMP1) and the non-coding sequence Pfr364 for P. falciparum were targeted to design new LAMP primers. The limit of detection (LOD) of each colorimetric LAMP was established and assessed with DNA extracted by mini spin column kit and the Boil & Spin method from 28 microscopy infections, 101 malaria submicroscopic infections detected by real-time PCR only, and 183 negatives infections by both microscopy and PCR.ResultsThe LODs for the colorimetric LAMPs were estimated between 2.4 to 3.7 parasites/µL of whole blood. For P. vivax detection, the colorimetric LAMP using the COX1 target showed a better performance than the Pvr47 target, whereas the Pfr364 target was the most specific for P. falciparum detection. All microscopic infections of P. vivax were detected by PvCOX1-LAMP using the mini spin column kit DNA extraction method and 81% (17/21) were detected using Boil & Spin sample preparation. Moreover, all microscopic infections of P. falciparum were detected by Pfr364-LAMP using both sample preparation methods. In total, PvCOX1-LAMP and Pfr364-LAMP detected 80.2% (81 samples) of the submicroscopic infections using the DNA extraction method by mini spin column kit, while 36.6% (37 samples) were detected using the Boil & Spin sample preparation method.ConclusionThe colorimetric LAMPs with multicopy targets using the COX1 target for P. vivax and the Pfr364 for P. falciparum have a high potential to improve POC malaria diagnosis detecting a greater number of submicroscopic Plasmodium infections.

Simple and rapid detection Aspergillus fumigatus by loop-mediated isothermal amplification coupled with lateral flow biosensor assay.

We have developed a new diagnostic technique, termed loop-mediated isothermal amplification coupled with lateral flow biosensor (LAMP-LFB), which has been successfully applied to the detection of Aspergillus fumigatus. A set of six LAMP primers was designed according to the A. fumigatus-specific anxC4 gene, which specifically recognized eight different regions of the target sequence. The LFB was employed for reporting the A. fumigatus-LAMP results, and the visual readouts were obtained within 2min. The strains of A. fumigatus species and non-A. fumigatus species were used to test the assay's sensitivity and examine the analytical specificity of the target assay. Optimal LAMP conditions were 66°C for 50min. The limit of detection is 100fg. No cross-reactions were obtained, and the specificity of LAMP-LFB assay was 100%. The whole process of the assay, including 20min of DNA preparation, 50min of constant temperature amplification, and 2min of detection by the sensor strip, took a total of 72min (less than 75min). Among 89 sputum specimens for clinical evaluation, 10 (11·23%) samples were A. fumigatus-positive by LAMP-LFB and traditional culture method, 9 (10·11%) samples were A. fumigatus-positive by PCR method. Compared with culture method, the diagnostic accuracy of LAMP-LFB method was 100%. The novel LAMP-LFB detection technology established in the current research is a rapid and reliable detection tool for A. fumigatus. This novel LAMP-LFB assay can quickly, specifically and sensitively detect A. fumigatus, thereby speeding up the detection process and increasing the detection rate. In addition, it can also be used as a new molecular method for detection of A. fumigatus in clinical and laboratory areas.

Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid and On-Site Detection of Avian Influenza Virus.

Avian influenza virus (AIV) outbreaks occur frequently worldwide, causing a potential public health risk and great economic losses to poultry industries. Considering the high mutation rate and frequent genetic reassortment between segments in the genome of AIVs, emerging new strains are a real threat that may infect and spread through the human population, causing a pandemic. Therefore, rapid AIV diagnostic tests are essential tools for surveillance and assessing virus spreading. Real-time reverse transcription PCR (rRT-PCR), targeting the matrix gene, is the main official standard test for AIV detection, but the method requires well-equipped laboratories. Reverse transcription Loop-Mediated Isothermal Amplification (RT-LAMP) has been reported as a rapid method and an alternative to PCR in pathogen detection. The high mutation rate in the AIV genome increases the risk of false negative in nucleic acid amplification methods for detection, such as PCR and LAMP, due to possible mismatched priming. In this study, we analyzed 800 matrix gene sequences of newly isolated AIV in the EU and designed a highly efficient LAMP primer set that covers all AIV subtypes. The designed LAMP primer set was optimized in real-time RT-LAMP (rRT-LAMP) assay. The rRT-LAMP assay detected AIV samples belonging to nine various subtypes with the specificity and sensitivity comparable to the official standard rRT-PCR assay. Further, a two-color visual detection RT-LAMP assay protocol was adapted with the aim to develop on-site diagnostic tests. The on-site testing successfully detected spiked AIV in birds oropharyngeal and cloacal swabs samples at a concentration as low as 100.8 EID50 per reaction within 30 minutes including sample preparation. The results revealed a potential of this newly developed rRT-LAMP assay to detect AIV in complex samples using a simple heat treatment step without the need for RNA extraction.

Point of care colourimetric and lateral flow LAMP assay for the detection of Haemonchus contortus in ruminant faecal samples.

In this study, we present an optimised colourimetric and a lateral flow LAMP assay for the detection of Haemonchus contortus in small ruminant faecal samples. Using a previously published LAMP primer set, we made use of commercially available colourimetric LAMP and lateral flow kits and combined this into an optimised diagnostic assay which was then tested on field faecal samples from Eastern and South-Eastern Hungary as well as a pure H. contortus egg faecal sample from Košice, Slovakia. Both assays showed no conflicts in visual detection of the results. Additionally, we modified and tested several centrifuge-free DNA extraction methods and one bead-beating egg lysis DNA extraction method to develop a true point of care protocol, as the source of the starting DNA is the main rate-limiting step in farm-level molecular diagnosis. Out of the various methods trialed, promising results were obtained with the magnetic bead extraction method. Sample solutions from the Fill-FLOTAC® technique were also utilised, which demonstrated that it could be efficiently adapted for field-level egg concentration to extract DNA. This proof of concept study showed that isothermal amplification technologies with a colourimetric detection or when combined with a lateral flow assay could be an important step for a true point of care molecular diagnostic assay for H. contortus.

Multiplex reverse transcription loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for the diagnosis of COVID-19

The ongoing global pandemic (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a huge public health issue. Hence, we devised a multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) coupled with a nanoparticle-based lateral flow biosensor (LFB) assay (mRT-LAMP-LFB) for diagnosing COVID-19. Using two LAMP primer sets, the ORF1ab (opening reading frame 1a/b) and N (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified in a single-tube reaction, and detected with the diagnosis results easily interpreted by LFB. In presence of FITC (fluorescein)-/digoxin- and biotin-labeled primers, mRT-LAMP produced numerous FITC-/digoxin- and biotin-attached duplex amplicons, which were determined by LFB through immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the test line of LFB) and biotin/treptavidin interaction (biotin on the duplex and strptavidin on the polymerase nanoparticle). The accumulation of nanoparticles leaded a characteristic crimson band, enabling multiplex analysis of ORF1ab and N gene without instrumentation. The limit of detection (LoD) of COVID-19 mRT-LAMP-LFB was 12 copies (for each detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. The analytical sensitivity of SARS-CoV-2 was 100% (33/33 oropharynx swab samples collected from COVID-19 patients), and the assay's specificity was also 100% (96/96 oropharynx swab samples collected from non-COVID-19 patients). The total diagnostic test can be completed within 1 h from sample collection to result interpretation. In sum, the COVID-19 mRT-LAMP-LFB assay is a promising tool for diagnosing SARS-CoV-2 infections in frontline public health field and clinical laboratories, especially from resource-poor regions.

Naked Eye Detection of α0 Thalassemia --SEA Type using Hydroxynaphthol Blue Loop-mediated Isothermal Amplification

BACKGROUND: Alpha-thalassemia is a major health problem for worldwide populations due to its recent large-scale global movements to many other parts of the world. The most effective method to prevent the spread of the worst form of α-thalassemia disorder, hemoglobin Bart’s hydrops fetalis, is to identify the genotypes of an α0 thalassemia carrier and then conduct subsequent genetic counseling.&#x0D; AIM: This research was aimed to develop a new method for the detection of α0 thalassemia (Southeast Asia or SEA) --SEA type using hydroxynaphthol blue loop-mediated isothermal amplification (HNB-LAMP) and by visually reading results using the naked eye.&#x0D; METHODS: This experimental research was conducted at Rangsit University, Pathum Thani Provinces, Thailand, from 2016 to 2018. Two sets of LAMP primers were used, one set of primer was used to detect the two existing α genes and the other set used in the detection of two α genes deletion --SEA types which were designed based on the nucleotide sequence of two α genes deletion from four cases of the α0 thalassemia carrier --SEA type. HNB-LAMP was developed, and then, 77 cases from subjects were evaluated by comparing them with a conventional polymerase chain reaction (PCR).&#x0D; RESULTS: The newly developed HNB-LAMP shows a high diagnostic sensitivity and diagnostic specificity, 100% in correct interpretation, from 10 cases of α0 thalassemia carriers --SEA type, 29 cases of α+ thalassemia carrier -α3.7 type, 8 cases of hemoglobin constant spring/hemoglobin Pakse′ (HbCS/HbPS), and 30 standard cases.&#x0D; CONCLUSION: This newly developed HNB-LAMP is suitable for in the field applications for detecting the presence of α0 thalassemia --SEA type due to its high diagnostic sensitivity, high diagnostic specificity, and, most notably, the ability to be detected with the naked eye.

Development of an FTP-LAMP assay based on TaqMan real-time PCR and LAMP for the specific detection of Xylella fastidiosa De Donno and mulberry strains in both plants and insect vectors

We developed two real-time detection assays, TaqMan real-time PCR and LAMP, using primers and probe designed based on a sequence annotated to code for a Haemagglutinin-related protein (Hg) of Xylella fastidiosa (Xf), a gene uniquely present in the Italian olive (De Donno of olive) and American mulberry strains, for specific detection of the target Xf strains. These assays were validated with DNA samples extracted from Xf-infected plant samples and from two species of insect vectors (Philaenus spumarius, Ps; and Neophilaenus campestris, Nc). Both techniques were proven to be highly sensitive (100 fg of Xf-genomic DNA) and specific to the Italian De Donno and American mulberry strains of Xf. When our LAMP was utilized in a duplex manner by combining with previously published universal primers and probe for detection of all Xf-subspecies and strains, the duplex LAMP showed high versatility in the simultaneous detection and differentiation of the Italian De Donno and American mulberry stains form other subspecies/strains. Furthermore, the Hg gene-specific LAMP primers and TaqMan probe were exploited to develop a new approach; henceforth referred to as the Fluorescence of TaqMan Probe upon Dequenching - Loop mediated Isothermal Amplification (FTP-LAMP). In the FTP-LAMP, the Xf-Hg specific fluorophore-quenched probe was added to a standard LAMP reaction and fluoresces only when bound to its target, allowing for a sequence-specific detection of the Xf-Italian De Donno and American mulberry strains in a LAMP context. Our FTP-LAMP assay showed to be highly sensitive detecting down to 100 fg genomic DNA of Xf, when tested on Xf-genomic DNA extracted from infected plants, DAS-ELISA-crude saps and insect vectors. Furthermore, the assay showed high specificity (98.7% vs 89% for LAMP) when applied on DNA templates from insect vectors. With the addition of an extra target sequence-specific probe acting as a direct Xf-specific dye, the FTP-LAMP has gained more specificity and reduced one of the main problems of the LAMP assay (false positives) when used for detecting of Xf in insect vectors. To the best of our knowledge, this study reports the development of the first LAMP assay and the first novel FTP-LAMP method for specific detection of the Italian De Donno and the American mulberry strains of Xf. Together with the Xf universal LAMP primers in a duplex approach, the FTP-LAMP could represent a useful tool not only for the specific detection of the olive-associated strain in Italy, but also to differentiate the De Donno strain from other strains of Xf already reported in Italy and Europe (Germany, France, Spain and Portugal).

Further development of a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of foot-and-mouth disease virus and validation in the field with use of an internal positive control.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hooved animals. Global outbreaks have highlighted the significant economic, trade, psychosocial and animal welfare impacts that can arise from the detection of disease in previously 'FMD-free' countries. Rapid and early diagnosis provides significant advantages in disease control and minimization of deleterious consequences. We describe the process of further development and validation of a reverse-transcription loop-mediated isothermal amplification foot-and-mouth disease virus (RT-LAMP-FMDV) test, using a published LAMP primer set, for use in the field. An internal positive control (IPC) was designed and introduced for use with the assay to mitigate any intrinsic interference from the unextracted field samples and avoid false negatives. Further modifications were included to improve the speed and operability of the test, for use by non-laboratory trained staff operating under field conditions, with shelf-stable reaction kits which require a minimum of liquid handling skills. Comparison of the assay performance with an established laboratory-based real-time reverse transcriptase PCR (rRT-PCR) test targeting the 3D region of FMD virus (Tetracore Inc) was investigated. LAMP has the potential to complement current laboratory diagnostics, such as rRT-PCR, as a preliminary tool in the investigation of FMD. We describe a strategic approach to validation of the test for use in the field using extracted RNA samples of various serotypes from Thailand and then finally unextracted field samples collected from FMD-suspected animals (primarily oral lesion swabs) from Bhutan and Australia. The statistical approach to validation was performed by Frequentist and Bayesian latent class methods, which both confirmed this new RT-LAMP-FMDV test as fit-for-purpose as a herd diagnostic tool with diagnostic specificity >99% and sensitivity 79% (95% Bayesian credible interval: 65, 90%) on unextracted field samples (oral swabs).