scholarly journals Combining LAMP and Au-Nanoprobe to detect INH-RIF resistance accurately in tuberculosis: an evidence-based review

2021 ◽  
Vol 15 (11) ◽  
pp. 1555-1568
Author(s):  
Muhammad Habiburrahman ◽  
Haekal Ariq ◽  
Raden Rara Diah Handayani
Keyword(s):  
Developing Countries ◽  
Color Change ◽  
New Technology ◽  
Accurate Method ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Evidence Based ◽  
Target Sequence ◽  
Loop Mediated Isothermal Amplification ◽  
Development Of Resistance

Approximately 1.41 million people die annually due to tuberculosis. One of the main problems in Tuberculosis eradication is the development of resistance to various antibiotics. However, current efforts to detect resistances face challenges such as limited equipment, budget, and time. This evidence-based review investigated loop-mediated isothermal amplification, an alternative molecular diagnostic tool with promising performance and applicability in developing countries, and its use combined with Au-Nanoprobe to detect antibiotic resistance in tuberculosis. The literature search was conducted through four databases (Proquest, EBSCOhost, Scopus, and Pubmed) for useful articles on loop-mediated isothermal amplification and Au-Nanoprobe in detecting tuberculosis and tuberculosis resistance. After filtering the result with inclusion and exclusion criteria, the search produced three papers that best answer the clinical question. Loop-mediated isothermal amplification amplifies a target sequence, and Au-Nanoprobe responds to the DNA specific to the target mutant, producing an observable color change. Loop-mediated isothermal amplification and Au-Nanoprobe showed 100% sensitivity and specificity in detecting rifampicin and isoniazid resistance. Another study investigated its viability to detect tuberculosis and found 98.2% sensitivity and 88.2% specificity. Combining loop-mediated isothermal amplification and Au-Nanoprobe had a shorter time to get results and should also be relatively cheaper because it does not need a high temperature to work and requires less equipment. In conclusion, loop-mediated isothermal amplification and Au-Nanoprobe can be used as an efficient and accurate method to detect isoniazid and rifampicin-resistant tuberculosis strains. The new technology is promising for developing countries due to their high disease burden but facing several healthcare barriers.

scholarly journals Development of a loop-mediated isothermal amplification (LAMP) method for specific detection of Mycobacterium bovis

2021 ◽  
Vol 15 (1) ◽  
pp. e0008996
Author(s):  
Thoko Flav Kapalamula ◽  
Jeewan Thapa ◽  
Mwangala Lonah Akapelwa ◽  
Kyoko Hayashida ◽  
Stephen V. Gordon ◽  
...  
Keyword(s):  
Developing Countries ◽  
Mycobacterium Bovis ◽  
Color Change ◽  
Low Cost ◽  
Lamp Assay ◽  
Genomic Region ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification

Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection of M. bovis is fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective against M. bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification of M. bovis by targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely in M. bovis. The assay's specificity was evaluated using 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven M. tuberculosis complex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected only M. bovis isolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies of M. bovis genomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection of M. bovis infections in cattle and humans in resource-limited areas.

scholarly journals 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 ◽  
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.

scholarly journals Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Bruna de Oliveira Coelho ◽  
Heloisa Bruna Soligo Sanchuki ◽  
Dalila Luciola Zanette ◽  
Jeanine Marie Nardin ◽  
Hugo Manuel Paz Morales ◽  
...  
Keyword(s):  
Viral Load ◽  
Internal Control ◽  
Reverse Transcription ◽  
Color Change ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
False Negative ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

Abstract Background SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. Methods To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. Results First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. Conclusion This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

scholarly journals Rapid detection of cytochrome cd1-containing nitrite reductase encoding gene nirS of denitrifying bacteria with loop-mediated isothermal amplification assay

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xuzhi Zhang ◽  
Qianqian Yang ◽  
Qingli Zhang ◽  
Xiaoyu Jiang ◽  
Xiaochun Wang ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Genomic Dna ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Denitrifying Bacteria ◽  
Isothermal Amplification ◽  
Target Sequence ◽  
Bacterial Cells ◽  
Loop Mediated Isothermal Amplification ◽  
Order Of Magnitude

Abstract The cytochrome cd1-containing nitrite reductase, nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance of nirS is a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a rapid method for detecting nirS gene with loop-mediated isothermal amplification (LAMP) was developed, using Pseudomonas aeruginosa PAO1 (P. aeruginosa PAO1) as model microorganism to optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, it was validated that P. aeruginosa PAO1 cells as well as genomic DNA could be directly used as template. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success. The nirS gene of P. aeruginosa PAO1 in spiked seawater samples could be detected with both DNA-template based LAMP assay and cell-template based LAMP assay, demonstrating the practicality of in-field use.

scholarly journals Loop-mediated isothermal amplification (LAMP) : A rapid molecular diagnosis technique for detection of human pathogens

2017 ◽  
Vol 07 (03) ◽  
pp. 042-048
Author(s):  
Gunimala Chakraborty ◽  
Indrani Karunasagar ◽  
Anirban Chakraborty
Keyword(s):  
Treatment Strategies ◽  
Lamp Assay ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Current Status ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Human Pathogens ◽  
Quality Healthcare ◽  
Loop Mediated Isothermal Amplification

AbstractDelivery of quality healthcare in case of an infectious disease depends on how efficiently and how quickly the responsible pathogens are detected from the samples. Molecular methods can detect the presence of pathogens in a rapid and sensitive manner. Over the years, a number of such assays have been developed. However, these methods, although highly reliable and efficient, require use of expensive equipment, reagents, and trained personnel. Therefore, development of molecular assays that are simple, rapid, cost-effective, yet sensitive, is highly warranted to ensure efficient management or treatment strategies. Loop-mediated isothermal amplification (LAMP), a technique invented in the year 2000, is a novel method that amplifies DNA at isothermal conditions. Since its invention, this technique has been one of the most extensively used molecular diagnostic tools in the field of diagnostics offering rapid, accurate and cost-effective diagnosis of infectious diseases. Using the LAMP principle, many commercial kits have been developed in the last decade for a variety of human pathogens including bacteria, viruses and parasites. Currently LAMP assay is being considered as an effective diagnostic tool for use in developing countries because of its simple working protocol, allowing even an onsite application. The focus of this review is to describe the salient features of this technique the current status of development of LAMP assays with an emphasis on the pathogens of clinical significance.

scholarly journals Monitoring Methyl Benzimidazole Carbamate-Resistant Isolates of the Cucurbit Powdery Mildew Pathogen, Podosphaera xanthii, Using Loop-Mediated Isothermal Amplification

Plant Disease ◽  
2019 ◽  
Vol 103 (7) ◽  
pp. 1515-1524 ◽  
Author(s):  
Alejandra Vielba-Fernández ◽  
Antonio de Vicente ◽  
Alejandro Pérez-García ◽  
Dolores Fernández-Ortuño
Keyword(s):  
Amino Acid ◽  
Powdery Mildew ◽  
Amino Acid Change ◽  
Color Change ◽  
Isothermal Amplification ◽  
Podosphaera Xanthii ◽  
Lamp Product ◽  
Loop Mediated Isothermal Amplification ◽  
Cucurbit Powdery Mildew ◽  
Β Tubulin

Powdery mildew, caused by the fungus Podosphaera xanthii, is one of the most economically important diseases affecting cucurbit crops in Spain. Currently, chemical control offers the most efficient management of the disease; however, P. xanthii isolates resistant to multiple classes of site-specific fungicides have been reported in the Spanish cucurbit powdery mildew population. In previous studies, resistance to the fungicides known as methyl benzimidazole carbamates (MBCs) was found to be caused by the amino acid substitution E198A on β-tubulin. To detect MBC-resistant isolates in a faster, more efficient, and more specific way than the traditional methods used to date, a loop-mediated isothermal amplification (LAMP) system was developed. In this study, three sets of LAMP primers were designed. One set was designed for the detection of the wild-type allele and two sets were designed for the E198A amino acid change. Positive results were only obtained with both mutant sets; however, LAMP reaction conditions were only optimized with primer set 2, which was selected for optimal detection of the E198A amino acid change in P. xanthii-resistant isolates, along with the optimal temperature and duration parameters of 65°C for 75 min, respectively. The hydroxynaphthol blue (HNB) metal indicator was used for quick visualization of results through the color change from violet to sky blue when the amplification was positive. HNB was added before the amplification to avoid opening the lids, thus decreasing the probability of contamination. To confirm that the amplified product corresponded to the β-tubulin gene, the LAMP product was digested with the enzyme LweI and sequenced. Our results show that the LAMP technique is a specific and reproducible method that could be used for monitoring MBC resistance of P. xanthii directly in the field.

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

BioTechniques ◽  
2020 ◽  
Vol 69 (3) ◽  
pp. 178-185 ◽  
Author(s):  
Yinhua Zhang ◽  
Guoping Ren ◽  
Jackson Buss ◽  
Andrew J Barry ◽  
Gregory C Patton ◽  
...  
Keyword(s):  
Guanidine Hydrochloride ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Simple Method ◽  
Diagnostic Assays ◽  
Loop Mediated Isothermal Amplification ◽  
Dna And Rna ◽  
Versatile Technique ◽  
Primer Sets

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.

scholarly journals Rapid detection of cytochromecd1-containing nitrite reductase encoding genenirSwith loop-mediated isothermal amplification assay

2018 ◽  
Author(s):  
Qianqian Yang ◽  
Xuzhi Zhang ◽  
Xiaoyu Jiang ◽  
Xiaochun Wang ◽  
Yang Li ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Genomic Dna ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Target Sequence ◽  
Bacterial Cells ◽  
Conventional Pcr ◽  
Loop Mediated Isothermal Amplification

AbstractThe cytochromecd1-containing nitrite reductase,nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance ofnirSis a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a new molecular biology technique termed loop-mediated isothermal amplification (LAMP) was developed to rapidly detectnirSgene using those ofPseudomonas aeruginosato optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The specificity of the assay was confirmed by the lack of amplification when using DNA from 15 other bacterial species lackingnirSgene. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, a cell-template based LAMP assay was also developed for detectingnirSgene that directly used bacterial cells as template rather than genomic DNA. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success, and bulky and sophisticated equipment were not needed. Further, thenirSgene ofP. aeruginosain spiked seawater samples could be detected with both the DNA-template based LAMP assay and the cell-template based LAMP assay, thereby demonstrating the practicality of in-field use of them. In summary, the LAMP assays described here represent a rapid, user-friendly, and cost-effective alternative to conventional PCR.

scholarly journals Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

2021 ◽  
Vol 12 ◽  
Author(s):  
Pedro A. Alves ◽  
Ellen G. de Oliveira ◽  
Ana Paula M. Franco-Luiz ◽  
Letícia T. Almeida ◽  
Amanda B. Gonçalves ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Isothermal Amplification ◽  
Alternative Methods ◽  
Molecular Diagnostic ◽  
Clinical Validation ◽  
Rna Detection ◽  
Loop Mediated Isothermal Amplification ◽  
Viral Transport Medium

The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 ± 2.7 viral genomic copies/μL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65°C for 30 min. When compared to reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3–99.5%] sensitivity and 100% (95% CI = 94.5–100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non–SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction–free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

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