Visual closed-tube loop-mediated isothermal amplification (LAMP) assay targeting aerolysin gene: a practical screening method for virulent Aeromonas species affecting cultured eels in China

To strengthen malaria surveillance, field-appropriate diagnostics requiring limited technical resources are of critical significance. Loop-mediated isothermal amplification (LAMP) based malaria diagnostic assays are potential point-of-care tests with high sensitivity and specificity and have been used in low-resource settings. Plasmodium vivax–specific consensus repeat sequence (CRS)-based and Plasmodium falciparum–specific 18S rRNA primers were designed, and a two-tube LAMP assay was developed. The diagnostic performance of a closed-tube LAMP assay and Loopamp™ Malaria Detection (Pan/Pf, Pv) kit was investigated using nested PCR confirmed mono- and co-infections of P. vivax and P. falciparum positive (n = 149) and negative (n = 67) samples. The closed-tube Pv LAMP assay showed positive amplification in 40 min (limit of detection, LOD 0.7 parasites/µL) and Pf LAMP assay in 30 min (LOD 2 parasites/µL). Pv LAMP and Pf LAMP demonstrated a sensitivity and specificity of 100% (95% CI, 95.96–100% and 89.85–100%, respectively). The LoopampTM Pan/Pf Malaria Detection kit demonstrated a sensitivity and specificity of 100%, whereas LoopampTM Pv showed a sensitivity of 98.36% (95% CI, 91.28–99.71%) and specificity of 100% (95% CI, 87.54–100%). The developed two-tube LAMP assay is highly sensitive (LOD ≤ 2 parasite/µL), demonstrating comparable results with the commercial Loopamp™ Malaria Detection (Pf/pan) kit, and was superior in detecting the P. vivax co-infection that remained undetected by the Loopamp™ Pv kit. The developed indigenous two-tube Pf/Pv malaria detection can reliably be used for mass screening in resource-limited areas endemic for both P. falciparum and P. vivax malaria.

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Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RTLAMP) for detecting SARS-Cov-2 in Clinical, Environmental and Animal Samples

University of the Future: Re-Imagining Research and Higher Education ◽

10.29117/quarfe.2020.0288 ◽

2020 ◽

Author(s):

Fatiha M. Benslimane ◽

Ola Al-Jamal ◽

Sonia Boughattas ◽

Asmaa A Al Than ◽

Hadi M. Yassine

Keyword(s):

New England ◽

Rna Extraction ◽

Screening Method ◽

Lamp Assay ◽

High Sensitivity ◽

Isothermal Amplification ◽

Molecular Diagnostic ◽

Reliable Technique ◽

Loop Mediated Isothermal Amplification ◽

Diagnostic Applications

Background: First described 20 years ago by Notomi et al. (1999), the loop-mediated isothermal amplification (LAMP) assay is robust, rapid and straightforward, yet retains high sensitivity and specificity. These features have seen the LAMP assay and the inclusion of a reverse transcriptase (RT-LAMP) implemented for a broad range of molecular diagnostic applications extending from infectious diseases, including detection of the original SARS-CoV-1 virus. The advantages of RTLAMP include using different reagents than RT-qPCR, the potential for direct processing of samples without the need for prior RNA extraction and an extremely rapid turn-around time. Several groups have now described different RT-LAMP assays for detection of SARS-CoV-2 RNA. Therefore, the aim of this study is to assess the feasibility, sensitivity and effectiveness of LAMP technique in detecting SARS-CoV-2 in different type of samples. Method: New England Biolabs (NEB) LAMP master mixes were used. Six set of primers specific to SARS-CoV-2 were obtained from IDT. The reaction mix consisting of LAMP master mix, primer working solution and a sample was incubated at 65⁰C and results were collected after 30 mins. Results: In just 30 mins, we were able to detect the virus without any prior sample processing. Our primers were able to detect up to 100 copies of the viruses, which is comparable to the RT-PCR that we currently use in our lab. The primers were tested against all other coronavirus and they have shown 100% specificity to the novel SARS-CoV-2 virus. Both the florescent and calorimetric master mixes were able to detect the virus in all tested samples: clinical, animal and environmental. Conclusion: LAMP is a fast reliable technique that could be used as a quick screening method for the detection of SARS-CoV-2 in different settings and using different collection medium.

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Development of a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of sweet potato latent virus-lotus in lotus plants

Australasian Plant Pathology ◽

10.1007/s13313-020-00714-8 ◽

2020 ◽

Vol 49 (4) ◽

pp. 425-429

Author(s):

Zhen He ◽

Tingting Dong ◽

Weiwen Wu ◽

Tielin Wang ◽

LiangJun Li

Keyword(s):

Sweet Potato ◽

Reverse Transcription ◽

Rapid Detection ◽

Lamp Assay ◽

Isothermal Amplification ◽

Latent Virus ◽

Loop Mediated Isothermal Amplification

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Establishment and validation of a loop-mediated isothermal amplification (LAMP) assay targeting the ttrRSBCA locus for rapid detection of Salmonella spp. in food

Food Control ◽

10.1016/j.foodcont.2021.107973 ◽

2021 ◽

pp. 107973

Author(s):

Antonia Kreitlow ◽

André Becker ◽

Ulrich Schotte ◽

Burkhard Malorny ◽

Madeleine Plötz ◽

...

Keyword(s):

Rapid Detection ◽

Lamp Assay ◽

Isothermal Amplification ◽

Salmonella Spp ◽

Loop Mediated Isothermal Amplification

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Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison

Scientific Reports ◽

10.1038/s41598-021-91098-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Somayyeh Sedaghatjoo ◽

Monika K. Forster ◽

Ludwig Niessen ◽

Petr Karlovsky ◽

Berta Killermann ◽

...

Keyword(s):

Test Performance ◽

Genomic Dna ◽

Lamp Assay ◽

Fungal Species ◽

Isothermal Amplification ◽

Genome Comparison ◽

Performance Study ◽

Loop Mediated Isothermal Amplification ◽

Tilletia Controversa ◽

Genomic Regions

AbstractTilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.

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Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

Scientific Reports ◽

10.1038/s41598-021-83371-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Severino Jefferson Ribeiro da Silva ◽

Keith Pardee ◽

Udeni B. R. Balasuriya ◽

Lindomar Pena

Keyword(s):

Reverse Transcription ◽

Rapid Detection ◽

Quantitative Polymerase Chain Reaction ◽

Rna Extraction ◽

Lamp Assay ◽

Isothermal Amplification ◽

Serum Samples ◽

Low Resource ◽

Loop Mediated Isothermal Amplification ◽

One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

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