A Rapid, Sensitive, Low-Cost Assay for Detecting Hydrogenotrophic Methanogens in Anaerobic Digesters Using Loop-Mediated Isothermal Amplification

Understanding how the presence, absence, and abundance of different microbial genera supply specific metabolic functions for anaerobic digestion (AD) and how these impact on gas production is critical for a long-term understanding and optimization of the AD process. The strictly anaerobic methanogenic archaea are essential for methane production within AD microbial communities. Methanogens are a phylogenetically diverse group that can be classified into three metabolically distinct lineages based on the substrates they use to produce methane. While process optimization based on physicochemical parameters is well established in AD, measurements that could allow manipulation of the underlying microbial community are seldom used as they tend to be non-specific, expensive, or time-consuming, or a combination of all three. Loop-mediated isothermal amplification (LAMP) assays combine a simple, rapid, low-cost detection technique with high sensitivity and specificity. Here, we describe the optimization of LAMP assays for the detection of four different genera of hydrogenotrophic methanogens: Methanoculleus, Methanothermobacter, Methanococcus, and Methanobrevibacter spp. By targeting archaeal elongation factor 2 (aEF2), these LAMP assays provide a rapid, low-cost, presence/absence indication of hydrogenotrophic methanogens that could be used as a real-time measure of process conditions. The assays were shown to be sensitive to 1 pg of DNA from most tested methanogen species, providing a route to a quantitative measure through simple serial dilution of samples. The LAMP assays described here offer a simple, fast, and affordable method for the specific detection of four different genera of hydrogenotrophic methanogens. Our results indicate that this approach could be developed into a quantitative measure that could provide rapid, low-cost insight into the functioning and optimization of AD and related systems.

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Simple, sensitive and cost-effective detection of wAlbB Wolbachia in Aedes mosquitoes, using loop mediated isothermal amplification combined with the electrochemical biosensing method

10.1101/2021.06.30.450550 ◽

2021 ◽

Author(s):

Pattamaporn Kittayapong ◽

Parinda Thayanukul ◽

Benchaporn Lertanantawong ◽

Worachart Sirawaraporn ◽

Surat Charasmongkolcharoen ◽

...

Keyword(s):

Low Cost ◽

High Sensitivity ◽

Cost Effective ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification ◽

Control Programs ◽

Electrochemical Biosensing ◽

Dna Strand Displacement ◽

Displacement Based ◽

Dna Strand

Background Wolbachia is an endosymbiont bacterium generally found in about 40% of insects, including mosquitoes, but it is absent in Aedes aegypti which is an important vector of several arboviral diseasesdengue, chikungunya, Zika, and yellow fever. The evidence that Wolbachia trans-infected Ae. aegypti mosquitoes lost their vectorial competence and became less capable of transmitting arboviruses to human hosts highlights the potential of using Wolbachia - based approaches for prevention and control of arboviral diseases. Recently, release of Wolbachia trans-infected Ae. aegypti has been deployed widely in many countries for the control of mosquito-borne viral diseases. Field surveillance and monitoring of Wolbachia presence in released mosquitoes is important for the success of these control programs. So far, a number of studies have reported the development of loop mediated isothermal amplification (LAMP) assays to detect Wolbachia in mosquitoes, but the methods still have some specificity issues. Methodology/Principal Findings We describe here the development of a LAMP combined with the DNA strand displacement-based electrochemical sensor (BIOSENSOR) method to detect wAlbB Wolbachia in trans-infected Ae . aegypti . Our developed LAMP primers were more specific to wAlbB detection than those of the previous published ones if the assays were conducted with low-cost and non-specific detecting dyes. The detection capacity of our LAMP technique was 3.8 nM and the detection limit reduced to 2.16 fM when combined with the BIOSENSOR. Our study demonstrates that the BIOSENSOR can also be applied as a stand-alone method for detecting Wolbachia ; and it showed high sensitivity when used with the crude DNA extracts of macerated mosquito samples without DNA purification. Conclusions/Significance Our results suggest that both LAMP and BIOSENSOR, either used in combination or stand-alone, are robust and sensitive. The methods have good potential for routine detection of Wolbachia in mosquitoes during field surveillance and monitoring of Wolbachia -based release programs, especially in countries with limited resources.

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SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

Frontiers in Medicine ◽

10.3389/fmed.2021.627679 ◽

2021 ◽

Vol 8 ◽

Author(s):

Alfredo Garcia-Venzor ◽

Bertha Rueda-Zarazua ◽

Eduardo Marquez-Garcia ◽

Vilma Maldonado ◽

Angelica Moncada-Morales ◽

...

Keyword(s):

Limit Of Detection ◽

Direct Detection ◽

Direct Method ◽

Rna Extraction ◽

Rna Isolation ◽

High Sensitivity ◽

High Specificity ◽

Isothermal Amplification ◽

Clinical Samples ◽

Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

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Loop-Mediated Isothermal Amplification for Detection ofStaphylococcus aureusin Dairy Cow Suffering from Mastitis

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/435982 ◽

2012 ◽

Vol 2012 ◽

pp. 1-5 ◽

Cited By ~ 9

Author(s):

Zhang Tie ◽

Wang Chunguang ◽

Wei Xiaoyuan ◽

Zhao Xinghua ◽

Zhong Xiuhui

Keyword(s):

Staphylococcus Aureus ◽

High Sensitivity ◽

High Specificity ◽

Isothermal Amplification ◽

Lamp Method ◽

Specific Primers ◽

Loop Mediated Isothermal Amplification ◽

Reaction Tube ◽

Specificity And Sensitivity ◽

Detectable Concentration

To develop a rapid detection method ofStaphylococcus aureususing loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of thenucgene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was1×102 CFU/mL and that of PCR was1×104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection ofStaphylococcus aureushas many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection ofStaphylococcus aureus.

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Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients

Journal of Medical Microbiology ◽

10.1099/jmm.0.47499-0 ◽

2008 ◽

Vol 57 (4) ◽

pp. 439-443 ◽

Cited By ~ 68

Author(s):

Basu Dev Pandey ◽

Ajay Poudel ◽

Tomoko Yoda ◽

Aki Tamaru ◽

Naozumi Oda ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Lamp Assay ◽

High Sensitivity ◽

Isothermal Amplification ◽

Nucleic Acid Amplification ◽

Lamp Method ◽

Rrna Gene ◽

Predictive Values ◽

Loop Mediated Isothermal Amplification ◽

System A

A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5–97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 %, respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.

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Loop Mediated Isothermal Amplification: Principles and Applications in Plant Virology

Plants ◽

10.3390/plants9040461 ◽

2020 ◽

Vol 9 (4) ◽

pp. 461 ◽

Cited By ~ 9

Author(s):

Stefano Panno ◽

Slavica Matić ◽

Antonio Tiberini ◽

Andrea Giovanni Caruso ◽

Patrizia Bella ◽

...

Keyword(s):

Plant Protection ◽

Point Of Care ◽

Low Cost ◽

Isothermal Amplification ◽

Nucleic Acid Amplification ◽

Plant Virology ◽

Food Industries ◽

Loop Mediated Isothermal Amplification ◽

Application Fields ◽

Agriculture And Food

In the last decades, the evolution of molecular diagnosis methods has generated different advanced tools, like loop-mediated isothermal amplification (LAMP). Currently, it is a well-established technique, applied in different fields, such as the medicine, agriculture, and food industries, owing to its simplicity, specificity, rapidity, and low-cost efforts. LAMP is a nucleic acid amplification under isothermal conditions, which is highly compatible with point-of-care (POC) analysis and has the potential to improve the diagnosis in plant protection. The great advantages of LAMP have led to several upgrades in order to implement the technique. In this review, the authors provide an overview reporting in detail the different LAMP steps, focusing on designing and main characteristics of the primer set, different methods of result visualization, evolution and different application fields, reporting in detail LAMP application in plant virology, and the main advantages of the use of this technique.

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Loop-Mediated Isothermal Amplification as Point-of-Care Diagnosis for Neglected Parasitic Infections

International Journal of Molecular Sciences ◽

10.3390/ijms21217981 ◽

2020 ◽

Vol 21 (21) ◽

pp. 7981

Author(s):

Catalina Avendaño ◽

Manuel Alfonso Patarroyo

Keyword(s):

Neglected Tropical Diseases ◽

Point Of Care ◽

Low Cost ◽

High Specificity ◽

World Health Organisation ◽

Isothermal Amplification ◽

World Health ◽

Parasitic Infections ◽

River Blindness ◽

Loop Mediated Isothermal Amplification

The World Health Organisation (WHO) has placed twenty diseases into a group known as neglected tropical diseases (NTDs), twelve of them being parasitic diseases: Chagas’ disease, cysticercosis/taeniasis, echinococcosis, food-borne trematodiasis, human African trypanosomiasis (sleeping sickness), leishmaniasis, lymphatic filariasis, onchocerciasis (river blindness), schistosomiasis, soil-transmitted helminthiasis (ascariasis, hookworm, trichuriasis), guinea-worm and scabies. Such diseases affect millions of people in developing countries where one of the main problems concerning the control of these diseases is diagnosis-based due to the most affected areas usually being far from laboratories having suitable infrastructure and/or being equipped with sophisticated equipment. Advances have been made during the last two decades regarding standardising and introducing techniques enabling diagnoses to be made in remote places, i.e., the loop-mediated isothermal amplification (LAMP) technique. This technique’s advantages include being able to perform it using simple equipment, diagnosis made directly in the field, low cost of each test and the technique’s high specificity. Using this technique could thus contribute toward neglected parasite infection (NPI) control and eradication programmes. This review describes the advances made to date regarding LAMP tests, as it has been found that even though several studies have been conducted concerning most NPI, information is scarce for others.

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HIGHLY SENSITIVE H7N9 AVIAN INFLUENZA VIRUS DETECTION USING REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION AND OLIGONUCLEOTIDE MICROARRAY

Taiwan Veterinary Journal ◽

10.1142/s1682648515500195 ◽

2015 ◽

Vol 41 (04) ◽

pp. 251-255

Author(s):

Lih-Chiann Wang ◽

Dean Huang

Keyword(s):

Avian Influenza ◽

Sensitivity And Specificity ◽

Reverse Transcription ◽

High Sensitivity ◽

Oligonucleotide Microarray ◽

Influenza Viruses ◽

Isothermal Amplification ◽

Detection Methods ◽

Loop Mediated Isothermal Amplification ◽

Viral Copy Number

H7N9 avian influenza viruses have circulated in the human population and poultry flocks in China since 2013. H7N9 virus monitoring is imperative in Taiwan due to the frequent contact between China and Taiwan. Traditional viral molecular detection methods using RT-PCR and sequencing are time- and labor-intensive, thus a simpler and cheaper method with high sensitivity and specificity is worth developing. We successfully detected human and wild bird H7N9 viruses in this study using reverse transcription loop-mediated isothermal amplification (RT-LAMP) and oligonucleotide microarray. The detection limit was as low as one viral copy number. The specific matching reaction between the templates and microarray probes during hybridization ensured the high detection effectiveness. The excellent sensitivity and specificity of the RT-LAMP-microarray makes it a powerful H7N9 surveillance approach in Taiwan.

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Combination of loop-mediated isothermal amplification and AuNP-oligoprobe colourimetric assay for pork authentication in processed-meat products

10.1101/2020.07.12.199091 ◽

2020 ◽

Author(s):

Pattanapong Thangsunan ◽

Sasithon Temisak ◽

Phattaraporn Morris ◽

Leonardo Rios-Solis ◽

Nuttee Suree

Keyword(s):

Limit Of Detection ◽

Meat Products ◽

High Sensitivity ◽

Cross Reactivity ◽

Isothermal Amplification ◽

Processed Meat ◽

High Concentration ◽

Food Authentication ◽

Loop Mediated Isothermal Amplification ◽

Halal Food

AbstractPork adulteration is a major concern for Muslims and Jews whose diets are restricted by religious beliefs, as well as those who are allergic to pork meat and its derivatives. Accurate pork authentication is of great importance to assist this demographic group of people in making decision on their product purchase. The aim of this study was to develop a new analytical method for pork authentication in processed-meat products based on a combination of loop-mediated isothermal amplification (LAMP) and AuNP-nanoprobe colourimetric assay. The LAMP conditions were first optimised to obtain the highest yield of amplified DNA products within the shortest time. Oligoprobe-functionalised AuNPs were then hybridised with LAMP-DNA amplicons, and subsequently challenged with MgSO4 at a high concentration to induce AuNP aggregation. In the presence of pork DNA, the colloidal AuNPs-probe remained unchanged in its red colour, which indicates the dispersion of AuNPs. In contrast, in the absence of pork DNA, the colour was changed to colourless as a result from the aggregation of AuNPs. The LAMP-AuNP-nanoprobe assay offers a high sensitivity with a limit of detection as low as 100 pg of pork DNA. The assay is highly specific to pork content without cross-reactivity with the other meat species tested. The assay developed herein can become a simple, inexpensive, precise, and rapid analytical tool for small laboratories or the general public interested in halal food authentication.

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Specific and sensitive, ready-to-use universal fungi detection by visual color using ITS1 loop-mediated isothermal amplification combined hydroxynaphthol blue

PeerJ ◽

10.7717/peerj.11082 ◽

2021 ◽

Vol 9 ◽

pp. e11082

Author(s):

Ilada Choopara ◽

Yothin Teethaisong ◽

Narong Arunrut ◽

Sudaluck Thunyaharn ◽

Wansika Kiatpathomchai ◽

...

Keyword(s):

Point Of Care ◽

Colorimetric Detection ◽

High Sensitivity ◽

Fungal Species ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification ◽

Fungal Isolates ◽

Simple Detection ◽

Hydroxynaphthol Blue ◽

Life Threatening

Being ubiquitous, fungi are common opportunistic pathogens to humans that can lead to invasive and life-threatening infections in immunocompromised individuals. Eukaryote-resembling cell membrane and filamentous branches make the fungal diagnosis difficult. This study therefore developed a ready-to-use ITS1 loop-mediated isothermal amplification combined with hydroxynaphthol blue (LAMP-HNB) for rapid, sensitive and specific colorimetric detection of universal fungi in all phyla. The ITS1 LAMP-HNB could identify every evolutionary phylum of fungi according to sequence analyses. We tested a total of 30 clinically relevant fungal isolates (representing three major human pathogenic phyla of fungi, namely Zygomycota, Ascomycota and Basidiomycota) and 21 non-fungal isolates, and the ITS1 LAMP-HNB properly identified all isolates, with a detection limit of as low as 4.6 ag (9.6 copies), which was identical to ITS1 and 18S rDNA PCR. The assays were also validated on the feasibility of point-of-care diagnostic with real food (dry peanuts, chili and garlics) and blood samples. Furthermore, the shelf life of our ready-to-use ITS1 LAMP activity (≥50%) was more than 40 days at 30 °C with 3–5% polyvinyl alcohol or glycerol additive. The results supported the ready-to-use ITS1 LAMP-HNB for simple detection of fungi contamination with high sensitivity in local and resource-constrained areas to prevent opportunistic fungal species infections.

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LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR DETECTION OF C. BURNETII IN BULGARIA

Trakia Journal of Sciences ◽

10.15547/tjs.2021.02.006 ◽

2021 ◽

Vol 19 (2) ◽

pp. 147-151

Author(s):

M. Kunchev ◽

V. Belcheva ◽

E. Grigorov

Keyword(s):

Polymerase Chain Reaction ◽

Sensitivity And Specificity ◽

Q Fever ◽

High Sensitivity ◽

Isothermal Amplification ◽

Chain Reaction ◽

Retrospective Diagnosis ◽

Loop Mediated Isothermal Amplification ◽

The World ◽

Polymerase Chain

Q fever, which is caused by Coxiella burnetii, a small, pleomorphic intracellular bacterium, is the most widespread zoonosis in the world. The chronic form of the disease can lead to disability and death. Rapid diagnosis of Q fever is needed in order that effective treatment can be initiated. The conventional retrospective diagnosis of Q fever, based on serology, is useless for the treatment of afflicted patients. Thus, molecular methods have been created to close the diagnostic gap between the onset of the disease and the presence of specific antibodies in serum. A polymerase chain reaction is a suitable and reliable method with high sensitivity and specificity, but it requires expensive equipment and post-amplification protocol. Loop-mediated isothermal amplification (LAMP) is an isothermal technique, conducted at constant temperature that can amplify a negligible amount of DNA to more than 109 copies within one hour, using special primers and polymerase. We have tested the sensitivity and specificity of LAMP in the detection of C. burnetii. The mean positive rate of LAMP and polymerase chain reaction in patients was 100% and 74%, respectively. LAMP reacted negatively with non-C. burnetii pathogens and non-infected blood samples. We conclude that LAMP is a sensitive and specific technique for the detection of C. burnetii and has advantages over serological methods and PCR that make it attractive for diagnosing Q fever in countries around the world.

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