Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride

Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated techniques and enabling accurate, high-throughput diagnostics.

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Enhancing Colorimetric LAMP Amplification Speed and Sensitivity with Guanidine Chloride

10.1101/2020.06.03.132894 ◽

2020 ◽

Cited By ~ 5

Author(s):

Yinhua Zhang ◽

Guoping Ren ◽

Jackson Buss ◽

Andrew J. Barry ◽

Gregory C. Patton ◽

...

Keyword(s):

Guanidine Hydrochloride ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Simple Method ◽

Diagnostic Assays ◽

Dna And Rna ◽

Target Dna ◽

Versatile Technique ◽

Guanidine Chloride ◽

Primer Sets

AbstractLoop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated technique and enabling accurate and high-throughput diagnostics.

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Loop-mediated isothermal amplification (LAMP) for detection of the red ring nematode, Bursaphelenchus cocophilus

Nematology ◽

10.1163/15685411-00003069 ◽

2017 ◽

Vol 19 (5) ◽

pp. 559-565 ◽

Cited By ~ 1

Author(s):

Tatsuya Ide ◽

Natsumi Kanzaki ◽

Pedro Pablo Parra Giraldo ◽

Robin M. Giblin-Davis

Keyword(s):

Large Subunit ◽

Isothermal Amplification ◽

Internal Transcribed Spacers ◽

Simple Method ◽

Loop Mediated Isothermal Amplification ◽

Third Stage ◽

Stage Juvenile ◽

Total Dna ◽

Primer Sets ◽

Rna Genes

As a first step in developing a quick, accurate and simple method for the diagnosis of red ring disease, the loop-mediated isothermal amplification (LAMP)-based identification procedure was applied to the causative agent,Bursaphelenchuscocophilus. Two LAMP primer sets were designed using two loci of ribosomal RNA genes,i.e., D2-D3 expansion segments of the large subunit (D2-D3 LSU), and internal transcribed spacers (ITS). Within those two sets of primers, the D2-D3 LSU primer set successfully yielded amplicons fromB. cocophilusnematode lysate prepared from 3-year-old DESS-fixed specimens. The specificity of the primers was examined using 18 species of confamilial Aphelenchoididae nematodes and primer sensitivity was tested using a diluted series ofB. cocophiluslysate. The primer set did not amplify the DNA from other aphelenchoidids, and sensitivity was achieved by ‘1:100 diluted’B. cocophilusDNA (roughly 1/1500 of total DNA from a single third-stage juvenile).

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Development of a Multiplex Loop-Mediated Isothermal Amplification Assay for Diagnosis of Plasmodium spp., Plasmodium falciparum and Plasmodium vivax

Diagnostics ◽

10.3390/diagnostics11111950 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1950

Author(s):

Woong Sik Jang ◽

Da Hye Lim ◽

YoungLan Choe ◽

Hyunseul Jee ◽

Kyung Chul Moon ◽

...

Keyword(s):

Internal Control ◽

Point Of Care ◽

Lamp Assay ◽

18S Rrna Gene ◽

Isothermal Amplification ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Rrna Gene ◽

Loop Mediated Isothermal Amplification

Malaria, caused by the parasite Plasmodium and transmitted by mosquitoes, is an epidemic that mainly occurs in tropical and subtropical regions. As treatments differ across species of malarial parasites, there is a need to develop rapid diagnostic methods to differentiate malarial species. Herein, we developed a multiplex malaria Pan/Pf/Pv/actin beta loop-mediated isothermal amplification (LAMP) to diagnose Plasmodium spp., P. falciparum, and P. vivax, as well as the internal control (IC), within 40 min. The detection limits of the multiplex malaria Pan/Pf/Pv/IC LAMP were 1 × 102, 1 × 102, 1 × 102, and 1 × 103 copies/µL for four vectors, including the 18S rRNA gene (Plasmodium spp.), lactate dehydrogenase gene (P. falciparum), 16S rRNA gene (P. vivax), and human actin beta gene (IC), respectively. The performance of the LAMP assay was compared and evaluated by evaluating 208 clinical samples (118 positive and 90 negative samples) with the commercial RealStar® Malaria S&T PCR Kit 1.0. The developed multiplex malaria Pan/Pf/Pv/IC LAMP assay showed comparable sensitivity (100%) and specificity (100%) with the commercial RealStar® Malaria S&T PCR Kit 1.0 (100%). These results suggest that the multiplex malaria Pan/Pf/Pv/IC LAMP could be used as a point-of-care molecular diagnostic test for malaria.

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Loop-mediated isothermal amplification: an effective method for express-diagnostics of cancer

Cancer Urology ◽

10.17650/1726-9776-2018-14-2-88-99 ◽

2018 ◽

Vol 14 (2) ◽

pp. 88-99 ◽

Cited By ~ 4

Author(s):

Yu. A. Makarova ◽

A. A. Zotikov ◽

G. A. Belyakova ◽

B. Ya. Alekseev ◽

M. Yu. Shkurnikov

Keyword(s):

Lamp Assay ◽

Isothermal Amplification ◽

Diagnostic Methods ◽

Nucleic Acid Amplification ◽

Molecular Diagnostic ◽

Specific Method ◽

Reaction Products ◽

Loop Mediated Isothermal Amplification ◽

Highly Sensitive ◽

One Step

The review is devoted to loop-mediated isothermal amplification (LAMP) – a novel molecular diagnostic method that has recently become increasingly popular. Unlike polymerase chain reaction, LAMP does not require thermal cycling; DNA or RNA amplification occurs at a constant temperature (about 65 °C) with 4 or 6 primers. This is a fast, highly-sensitive, and highly specific method, which does not require expensive equipment, where visual detection of the reaction products is performed by the unaided eye. LAMP is successfully used for the diagnosis of multiple viruses, bacteria, and other pathogens (including those in food). Moreover, it can be applied for the detection of singlenucleotide polymorphisms. Recently, a modified LAMP assay – one-step nucleic acid amplification (OSNA) – was validated for metastasis detection. OSNA was demonstrated to have almost the same sensitivity and specificity as standard diagnostic methods (sometimes even higher). Particular attention is paid to the mechanism of LAMP, primer design, and diagnostics of cancer using OSNA.

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Loop-mediated isothermal amplification assay forEnterococcus sp., E. coliandS. aureusin chicken

10.1101/305805 ◽

2018 ◽

Author(s):

HyeSoon Song ◽

YouChan Bae ◽

HyukMan Kwon ◽

YongKuk Kwon ◽

SeongJoon Joh

Keyword(s):

Broiler Chicken ◽

Simultaneous Detection ◽

Isothermal Amplification ◽

Diagnostic Methods ◽

Economic Losses ◽

Diagnostic Assays ◽

E Coli ◽

Loop Mediated Isothermal Amplification ◽

Resource Limited ◽

Amplification Assay

ABSTRACTBacterial chondronecrosis with osteomyelitis (BCO) is a major cause of lameness in broiler chicken, and results in serious economic losses worldwide. Although the pathogenesis mechanism leading to lameness is not entirely understood, some strains ofEnterococcus sp., avian pathogenicEscherichia coli, orStaphylococcus aureushave been long recognized as important causative pathogens. To prevent the progression ofEnterococcus sp., avian pathogenicE. coli, orS. aureusinfections, we developed rapid, sensitive, and convenient diagnostic assays using loop-mediated isothermal amplification (LAMP). Entero-Common-LAMP assays were developed for a simultaneous detection of eightEnterococcusspecies. To target specific microorganisms, seven Entero-Specific-LAMP assays forE. faecalis, E. faecium, E. hirae, E. gallinarum, E. avium, E. duransandE. cecorum, andE. coli-LAMP andS. aureus-LAMP assays, were developed. Considering the prevalence and economic impact ofEnterococcus sp., E. coli, andS. aureus, the developed ten different LAMP assays have a considerable potential as routine diagnostic methods in the field or in resource-limited environments.

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A novel loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) device for rapid detection of Toxoplasma gondii in the blood of stray cats and dogs

Parasite ◽

10.1051/parasite/2021039 ◽

2021 ◽

Vol 28 ◽

pp. 41

Author(s):

Yangji Xue ◽

Qingming Kong ◽

Haojie Ding ◽

Chengzuo Xie ◽

Bin Zheng ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Lateral Flow ◽

Isothermal Amplification ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Diagnostic Tools ◽

Loop Mediated Isothermal Amplification ◽

Stray Cats ◽

Lateral Flow Dipstick ◽

Parasitic Pathogens

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis and threatens warm-blooded animal and human health worldwide. Simple and applicable diagnostic methods are urgently needed to guide development of effective approaches for prevention of toxoplasmosis. Most molecular diagnostic tools for T. gondii infection require high technical skills, sophisticated equipment, and a controlled lab environment. In this study, we developed a loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) assay that specifically targets the 529 bp for detecting T. gondii infection. This novel portable device is universal, fast, user-friendly, and guarantees experimental sensitivity as well as low risk of aerosol contamination. Our LAMP-LFD assay has a detection limit of 1 fg of T. gondii DNA, and shows no cross-reaction with other parasitic pathogens, including Cryptosporidium parvum, Leishmania donovani, and Plasmodium vivax. We validated the developed assay by detecting T. gondii in DNA extracted from blood samples collected from 318 stray cats and dogs sampled from Deqing, Wenzhou, Yiwu, Lishui and Zhoushan cities across Zhejiang province, Eastern China. The LAMP-LFD device detected T. gondii DNA in 4.76 and 4.69% of stray cats and dogs, respectively. In conclusion, the developed LAMP-LFD assay is efficient, minimizes aerosol contamination, and is therefore suitable for detecting T. gondii across basic medical institutions and field settings.

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Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass

3 Biotech ◽

10.1007/s13205-020-02602-w ◽

2021 ◽

Vol 11 (2) ◽

Author(s):

Domenico Rizzo ◽

Nicola Luchi ◽

Daniele Da Lio ◽

Linda Bartolini ◽

Francesco Nugnes ◽

...

Keyword(s):

Lamp Assay ◽

Isothermal Amplification ◽

Molecular Diagnostic ◽

Diagnostic Tools ◽

Loop Mediated Isothermal Amplification ◽

Larval Stages ◽

Longhorn Beetle ◽

Rapid Monitoring ◽

Major Pest ◽

Wood Borer

AbstractThe red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

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Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification

International Journal of Molecular Sciences ◽

10.3390/ijms21228741 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8741

Author(s):

Güven Edgü ◽

Lena Julie Freund ◽

Stefanie Hartje ◽

Eckhard Tacke ◽

Hans-Reinhard Hofferbert ◽

...

Keyword(s):

Disease Management ◽

Management Practices ◽

Tobacco Rattle Virus ◽

Simultaneous Detection ◽

Isothermal Amplification ◽

Diagnostic Methods ◽

Tuber Quality ◽

Loop Mediated Isothermal Amplification ◽

Tuber Necrosis ◽

Corky Ringspot

Potato is an important staple food crop in both developed and developing countries. However, potato plants are susceptible to several economically important viruses that reduce yields by up to 50% and affect tuber quality. One of the major threats is corky ringspot, which is a tuber necrosis caused by tobacco rattle virus (TRV). The appearance of corky ringspot symptoms on tubers prior to commercialization results in ≈ 45% of the tubers being downgraded in quality and value, while ≈ 55% are declared unsaleable. To improve current disease management practices, we have developed simple diagnostic methods for the reliable detection of TRV without RNA purification, involving minimalized sample handling (mini), subsequent improved colorimetric loop-mediated isothermal amplification (LAMP), and final verification by lateral-flow dipstick (LFD) analysis. Having optimized the mini-LAMP-LFD approach for the sensitive and specific detection of TRV, we confirmed the reliability and robustness of this approach by the simultaneous detection of TRV and other harmful viruses in duplex LAMP reactions. Therefore, our new approach offers breeders, producers, and farmers an inexpensive and efficient new platform for disease management in potato breeding and cultivation.

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Detection and discrimination of multiple strains of Zika virus by reverse transcription-loop-mediated isothermal amplification

Tropical Medicine and Health ◽

10.1186/s41182-020-00274-z ◽

2020 ◽

Vol 48 (1) ◽

Author(s):

Hiroka Aonuma ◽

Itoe Iizuka-Shiota ◽

Tokio Hoshina ◽

Shigeru Tajima ◽

Fumihiro Kato ◽

...

Keyword(s):

Virus Strain ◽

Reverse Transcription ◽

Zika Virus ◽

Virus Disease ◽

Isothermal Amplification ◽

Lamp Method ◽

Simple Method ◽

Loop Mediated Isothermal Amplification ◽

Specificity And Sensitivity ◽

Virus Strains

Abstract Background Monitoring both invasion of Zika virus disease into free countries and circulation in endemic countries is essential to avoid a global pandemic. However, the difficulty lies in detecting Zika virus due to the large variety of mutations in its genomic sequence. To develop a rapid and simple method with high accuracy, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was adopted for the detection of Zika virus strains derived from several countries. Results Common primers for RT-LAMP were designed based on the genomic sequences of two standard Zika strains: African lineage, MR-766, and Asian lineage, PRVABC59. RT-LAMP reactions using a screened primer set, targeting the NS3 region, detected both Zika virus strains. The minimum detectable quantity was 3 × 10−2 ng of virus RNA. Measurable lag of reaction times among strains was observed. The RT-LAMP method amplified the target virus sequence from the urine and serum of a patient with a travel history in the Caribbean Islands and also provided a prediction about which lineage of Zika virus strain was present. Conclusions The RT-LAMP method using a well-optimized primer set demonstrated high specificity and sensitivity for the detection of Zika virus strains with a variety in genomic RNA sequences. In combination with the simplicity of LAMP reaction in isothermal conditions, the optimized primer set established in this study may facilitate rapid and accurate diagnosis of Zika fever patients with virus strain information.

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Comparison of Loop-Mediated Isothermal Amplification and Real-Time PCR Assays for Detection ofStrongyloidesLarvae in Different Specimen Matrices

Journal of Clinical Microbiology ◽

10.1128/jcm.01173-18 ◽

2019 ◽

Vol 57 (4) ◽

Cited By ~ 1

Author(s):

Matthew R. Watts ◽

Rady Kim ◽

Vishal Ahuja ◽

Gemma J. Robertson ◽

Yasmin Sultana ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Chronic Infection ◽

Lamp Assay ◽

Isothermal Amplification ◽

Diagnostic Methods ◽

Percentage Agreement ◽

Content Type ◽

Loop Mediated Isothermal Amplification ◽

Stool Specimens

ABSTRACTStrongyloides stercoraliscan cause disease that ranges from asymptomatic chronic infection to fatal hyperinfection. Diagnosis from stool can be challenging because the most sensitive conventional tests require live larvae to be effective and there can be low larval output in chronic infection. Nucleic acid amplification tests (NAAT) have been developed to complement existing diagnostic methods. We compared a recently developed loop-mediated isothermal amplification (LAMP) assay with a real-time PCR that has previously been validated with larval microscopy. The limits of detection—quantified using serial dilutions of DNA extracts from singleStrongyloides rattithird-stage (L3) larvae spiked into approximately 250 µl of 5 differentS. stercoralis-negative stool specimens—were 10−3(1/5 replicates) and 10−2(1/5 replicates) dilutions for PCR and LAMP, respectively. PCR was positive for 4/5 replicates at 10−2. LAMP was compared to PCR using extracts from 396 stool specimens collected in Bangladesh and Australia, of which 53 were positive and 343 were negative by PCR. The positive percentage agreement of LAMP was 77.3% (95% score confidence interval [CI], 64.5 to 86.6). The negative percentage agreement was 100% (95% CI, 98.9 to 100). In a preliminary investigation, PCR and LAMP assays were positive using DNA extracted from serum (PCR, 3/16 extracts; LAMP, 2/16 extracts) and bronchoalveolar lavage fluid (PCR and LAMP, 2/2 extracts), demonstrating proof of concept. Compared to PCR, the lower number of positive results using the LAMP assay may have been due to reaction inhibitors and DNA degradation, and strategies to improve the LAMP assay are discussed.

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