Isothermal Amplification Technique Research Articles

Accurate and rapid detection of Fasciola hepatica copro‐DNA in sheep using loop‐mediated isothermal amplification (LAMP) technique

Fascioliasis is a parasitic infection caused by Fasciola spp. in humans and animals. Despite significant advances in vaccination and new therapeutic agents, little attention has been paid to validate methods for the diagnosis of fascioliasis in animals. This study aimed to compare the loop‐mediated isothermal amplification (LAMP) technique with PCR assay for the diagnosis of F. hepatica in sheep. In this cross‐sectional study, 195 stool samples were collected from sheep for 3 months in Lorestan province, West of Iran. Specimens’ parasitological examination was performed by using the direct wet mount and formalin‐ether concentration method. After DNA extraction from the samples, molecular analysis was done using PCR and LAMP techniques based on the Fasciola ribosomal intergenic spacer (IGS) sequence. Of 195 specimens of sheep, 11 specimens were identified as F. hepatica‐positive infection by using microscopic, PCR and LAMP assays. Kappa agreement test results showed that there was a significant agreement between the results of microscopic examination diagnostic tests, PCR and LAMP (Kappa = 0.51–0.72 and p < .001). According to the results of chi‐square comparisons between parasite prevalence applying different techniques and variables of age, sex breed, and type of drinking water, there was no significant relationship (p ≥ .05). However, most of the infected sheep with Fasciola were 3‐ to 4‐year‐old females, of the Lori breed and consumed tap water. In many endemic areas, successful prevention and treatment of fascioliasis in animals depend on rapid and accurate diagnosis. Based on the results of the Kappa agreement, the significant agreement among the results of the microscopic examination, PCR and LAMP indicates the accuracy and reliability of these tests in the diagnosis of F. hepatica in sheep. However, molecular methods, especially the LAMP technique, are suggested because of their higher sensitivity and reliability for the diagnosis of F. hepatica even under field conditions.

A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions

Isothermal nucleic acid amplification techniques are used to detect a variety of molecules such as RNAs, DNAs and proteins. When compared to the gold standard polymerase chain reaction, isothermal methods are rapid and simple but often have lower specificity and sensitivity. In this review, we provide a summary of reaction additives and techniques that enhance the sensitivity, specificity, stability, robustness, or speed of isothermal nucleic acid amplification reactions used for sensing applications. We also provide the mechanism and purpose of each enhancement technique, which can provide guidance when selecting reaction additives to optimize novel isothermal amplification systems and assays.

Isothermal Amplification of Nucleic Acids Coupled with Nanotechnology and Microfluidic Platforms for Detecting Antimicrobial Drug Resistance and Beyond.

Antibiotic resistance is one of the serious health-threatening issues globally, the control of which is indispensable for rapid diagnosis and treatment because of the high prevalence and risks of pathogenicity. Traditional and molecular techniques are relatively expensive, complex, and non-portable, requiring facilities, trained personnel, and high-tech laboratories. Widespread and timely-detection is vital to the better crisis management of rapidly spreading infective diseases, especially in low-tech regions and resource-limited settings. Hence, the need for inexpensive, fast, simple, mobile, and accessible point-of-care (POC) diagnostics is highly demanding. Among different biosensing methods, the isothermal amplification of nucleic acids is favorite due to their simplicity, high sensitivity/specificity, rapidity, and portability, all because they require a constant temperature to work. Isothermal amplification methods are utilized for detecting various targets, including DNA, RNA, cells, proteins, small molecules, ions, and viruses. In this paper, we discuss various platforms, applications, and potentials of isothermal amplification techniques for biosensing of antimicrobial resistance. We also evaluate the potential of these methods, coupled with the novel and rapidly-evolving platforms offered by nanotechnology and microfluidic devices.

Molecular evaluation of Toxocara species in stray cats using loop-mediated isothermal amplification (lamp) technique as a rapid, sensitive and simple screening assay.

Toxocara species are parasitic nematodes of dogs and cats with a worldwide distribution. The adult worm lives in the intestine, and horizontal transmission of the infection occurs through eating paratenic host or embryonated eggs. This study aimed to estimate the molecular prevalence of Toxocara species in stray cats using the loop‐mediated isothermal amplification (LAMP) technique. A total of 95 stool samples were randomly collected from stray cats in Khorramabad city in western Iran. Microscopic examination was performed after the separation and extraction of supernatants. The LAMP reaction was performed using the internal transcribed spacer 2 (ITS2) gene primers of Toxocara species and the appropriate master mix. The overall prevalence of Toxocara spp. in stray cats was 20% (19/95, CI 95%: 0.2 ± 0.08) by parasitological and molecular assessments. The microscopic examination of stool samples revealed that 19 samples were positive for Toxocara. The same 19 positive samples were also positive by the LAMP technique. Interestingly, based on the results of the LAMP assay, out of 95 studied samples, 18 (18.94%; CI 95%: 0.19 ± 0.08) specimens were Toxocara canis, while only 1 (1.05%; CI 95%: 0.005 ± 0.01) sample was diagnosed as Toxocara cati. The relatively high prevalence of Toxocara species in the studied cats shows the role of this species in spreading the parasite and the role of the cats in transmitting this zoonotic parasite. Preventive measures including the control of stray cat's population by castration and protection of public gardens where children play are recommended. The easy, highly sensitive and specific LAMP method is proposed for the differential detection of Toxocara species in animals and humans.

Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring.

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.

An isothermal amplification-based point-of-care diagnostic platform for the detection of Mycobacterium tuberculosis: A proof-of-concept study.

The timely diagnosis of active tuberculosis disease (TB) is crucial to interrupt the transmission and combat the spread of Mycobacterium tuberculosis (Mtb), the causative agent for TB. Here, we demonstrate the development of a specimen-direct rapid diagnostic method for TB which consists of an isothermal amplification device, Tiny Isothermal Nucleic acid quantification sYstem (TINY), coupled with helicase-dependent amplification (HDA). HDA, an isothermal amplification technique is established over TINY using pUCIDT-AMP vector carrying IS6110, the target DNA sequence for Mtb. The limit of detection of this technique for detecting the IS6110 within a threshold time of 50 min is 2.5 × 105 copies of IS6110. HDA in TINY for TB detection was evaluated using three IS6110-positive Mtb strains – H37Rv, CDC 1551, and Erdman wild-type and one IS6110-negative Mycobacterium avium. For spiked oral swabs, HDA in TINY detects IS6110 without any non-specificity in relatively short turnaround time (<1.5 h), highlighting its potential utility as a specimen-direct point-of-care diagnostic for TB. TINY does not require an uninterrupted power supply and its lightweight and small footprint offers portability and easier operation in clinical settings with poor infrastructure. Overall, HDA in TINY could serve as an efficient rapid, and portable platform for the qualitative detection of TB at the point-of-care.

Rapid genotyping of bacterial leaf blight resistant genes of rice using loop-mediated isothermal amplification assay.

The use of resistant (R) genes is the most effective strategy to manage bacterial leaf blight (BLB) disease of rice. Several attempts were made to incorporate R genes into susceptible rice cultivars using marker-assisted backcross breeding (MABB). However, MABB relies exclusively on PCR for foreground selection of R genes, which requires expensive equipment for thermo-cycling and visualization of results; hence, it is limited to sophisticated research facilities. Isothermal nucleic acid amplification techniques such as loop-mediated isothermal amplification (LAMP) assay do not require thermo-cycling during the assay. Therefore, it will be the best alternative to PCR-based genotyping. In this study, we have developed a LAMP assay for the specific and sensitive genotyping of seven BLB resistance (R) genes viz., Xa1, Xa3, Xa4, Xa7, Xa10, Xa11, and Xa21 in rice. Gene-specific primers were designed for the LAMP assay. The LAMP assay was optimized for time, temperature, and template DNA concentration. For effective detection, incubation at 60°C for 30min was optimum for all seven R genes. A DNA intercalating dye ethidium bromide and a calorimetric dye hydroxynaphthol blue was used for result visualization. Further, sensitivity assay revealed that the LAMP assay could detect R genes at 100fg of template DNA compared to 1ng and 10pg, respectively, in conventional PCR and q-PCR assays. The LAMP assay developed in this study provides a simple, specific, sensitive, robust, and cost-effective method for foreground selection of R genes in the resistance breeding programs of resource-poor laboratory.

Optimization of Loop-Mediated Isothermal Amplification (LAMP) reaction mixture for biosensor applications

Genetically Modified (GM) foods are becoming the future of agriculture on surviving global natural disasters and climate change by their enhanced production efficiency and improved functional properties. On the other hand, their adverse health and environmental effects, ample evidence on transgene leakage of Genetically Modified Organisms (GMOs) to crops have raised questions on their benefits and risks. Consequently, low-cost, reliable, rapid, and practical detection of GMOs have been important. GMO-detection platforms should be capable of stably storing detection reagents for long-delivery distances with varying ambient temperatures. In this study, we developed an event-specific, closed tube colorimetric GMO detection method based on Loop-Mediated Isothermal Amplification (LAMP) technique which can be integrated into GMO-detection platforms. The entire detection process optimized to 30 min and isothermally at 65 °C. The durability of the LAMP mixture in the test tubes showed that the LAMP reaction mixture, in which Bst polymerase and DNA sample was later included, yielded DNA amplicons for 3 days at room temperature, and for 6 days at 4 °C.•Simple, stable, and cheap storage method of LAMP reaction mixture for GMO-detection technologies.•GMO-detection platforms can stably store detection reagents for long-delivery distances with varying ambient temperatures.•Any DNA sample can be used in the field or resource-limited setting by untrained personnel.

Single tube amplification and detection of male date palm using polymerase chain reaction and loop-mediated isothermal amplification technique

Single tube amplification and detection of male date palm using polymerase chain reaction and loop-mediated isothermal amplification technique

A real-time LAMP-based dual-sample microfluidic chip for rapid and simultaneous detection of multiple waterborne pathogenic bacteria from coastal waters.

Waterborne pathogens are becoming a serious worldwide health hazard; thus, the regular monitoring of epidemic pathogens is urgently required for public safety. In the present study, we developed a microfluidic chip integrated loop-mediated isothermal amplification technique (on-chip LAMP) to simultaneously detect 10 waterborne pathogenic bacteria, Campylobacter jejuni, Listeria monocytogenes, Salmonella enterica, Shigella flexneri, Staphylococcus aureus, Vibrio alginolyticus, V. cholerae, V. parahemolyticus, V. vulnificus, and Yersinia enterocolitica. This method was capable of simultaneously completing 22 genetic analyses of two specimens and achieved limits of detection ranging from 7.92 × 10-3 to 9.54 × 10-1 pg of genomic DNA of pure bacteria per reaction. The processes from sample loading to microfluidic operation were in a highly automated format, and the LAMP reaction ran to completion within 35 minutes, with a minimal volume of 22 μl per each half of a single chip. The coefficient of variation for the time-to-positive value was less than 0.1, indicating an excellent reproducibility of the dual-sample on-chip LAMP assay. The clinical sensitivity and specificity in analyses of coastal water samples were 93.1% and 98.0%, respectively, in comparison with traditional microbiological methods. Our established dual-sample on-chip LAMP assay provides an effective multiple-pathogen analysis of waterborne bacterial pathogens. This indicates that the method is applicable for on-site detection and routine monitoring of waterborne bacteria in aquatic environments.

Direct Detection of SARS-CoV-2 Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Without RNA Purification

Coronavirus disease 2019 (COVID-19) is an acute respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which has spread globally. Shortage of nucleic acid extraction kits may delay the speed of diagnosis. In order to address this need, we propose a simple method for direct RNA extraction from nasopharyngeal swab samples without RNA purification. Here, we describe a reverse transcription loop-mediated isothermal amplification technique with 95.4% sensitivity (95% CI: 84.2-99.4%) and 100% specificity (95% CI: 78-100%). Combined with a simple RNA extraction step, this optimized RT-LAMP method is able to amplify SARS-CoV-2 directly from clinical nasopharyngeal swab samples in viral transport media in 50 minutes. This method provides an opportunity to facilitate SARS-CoV-2 detection especially in resource limited settings where nucleic acid extraction kits are few.Funding: This study was supported by Prototype Research Grant Scheme (PRGS), PR001-2020B (PRGS/2/2020/SKK09/UM/02/1) from the Ministry of Education, Malaysia.Declaration of Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.Ethics Approval Statement: Ethical approvals for this study was obtained from UMMC 85 Medical Ethics Committee (202041-8418) and Medical Research Ethics Committee 86 (MREC) Ministry of Health Malaysia (NMRR-20-2344-56994).

Single-Cell Analysis of Highly Metastatic Circulating Tumor Cells by Combining a Self-Folding Induced Release Reaction with a Cell Capture Microchip.

Nondestructive analysis of the single-cell molecular phenotype of circulating tumor cells (CTCs) is of great significance to the precise diagnosis and treatment of cancer but is also a huge challenge. To address this issue, here, we develop a facile analysis system that integrates CTCs' capture and molecular phenotype analysis. An isothermal nucleic acid amplification technique named self-folding induced release reaction (sFiR), which has high-efficiency signal amplification capabilities and can run under physiological conditions, is first developed to meet the high requirements for sensitivity and nondestructivity. By combining the sFiR with immune recognition and a single cell capture microchip, the molecular phenotype analysis of a single CTC is realized. As a model, nondestructive analysis of junction plakoglobin (JUP), an overexpressed membrane protein that is closely related to the metastasis of CTCs, is successfully achieved. Results reveal that this sFiR-based analysis system can clearly distinguish the expression of JUP in different cancer cell lines and can present quantitative information on the expression of JUP. Furthermore, the captured and analyzed CTCs maintain their basic physiological activity and can be used for drug sensitivity testing. Considering the excellent performance and ease of operation of the system, it can provide technical support for CTC-based cancer liquid biopsy and drug development.

Evaluation of different target genes for the detection of Salmonella sp. by loop‐mediated isothermal amplification

The loop-mediated isothermal amplification (LAMP) technique was used to investigate six salmonella-specific sequences for their suitability to serve as targets for the pathogen identification. Sequences selected for designing LAMP primers were genes invA, bcfD, phoP, siiA, gene62181533 and a region within the ttrRSBCA locus. Primers including single nucleotide polymorphisms were configured as degenerate primers. Specificity of the designed primer sets was determined by means of 46 salmonella and 32 other food- and waterborne bacterial reference species and strains. Primers targeting the ttrRSBCA locus showed 100 % inclusivity of target and exclusivity of other test species and strains. Other primer sets revealed deficiencies, especially regarding Salmonella enterica subsp. II-IV and Salmonella bongori. Additionally, primers targeting the siiA gene failed to detect S. enterica subsp. enterica serotypes Newport and Stanley, whereas bcfD primers did not amplify DNA of S. enterica subsp. enterica serotype Schleissheim. TtrRSBCA primers, providing short detection times and constant melting temperatures of amplification products, achieved best overall performance.

Combinations of PCR and Isothermal Amplification Techniques Are Suitable for Fast and Sensitive Detection of SARS-CoV-2 Viral RNA.

The newly identified coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causes coronavirus disease 2019 (COVID-19) and has affected over 25 million people worldwide as of August 31, 2020. To aid in the development of diagnostic kits for rapid and sensitive detection of the virus, we evaluated a combination of polymerase chain reaction (PCR) and isothermal nucleic acid amplification techniques. Here, we compared conventional PCR and loop-mediated isothermal amplification (LAMP) methods with hybrid techniques such as polymerase chain displacement reaction (PCDR) and a newly developed PCR-LAMP method. We found that the hybrid methods demonstrated higher sensitivity and assay reaction rates than those of the classic LAMP and PCR techniques and can be used to for SARS-CoV-2 detection. The proposed methods based on the modern hybrid amplification techniques markedly improve virus detection and, therefore, can be extremely useful in the development of new diagnostic kits.

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

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.

Identification of sheep (Ovis aries) meat by alkaline lysis-loop mediated isothermal amplification technique targeting mitochondrial D-loop region.

Identification of meat species origin using reliable techniques is a critical requirement for ensuring label compliance, protection of consumer preference and prevention of fraudulence in the meat trade. Although a plethora of protein and DNA based meat species identification techniques are in vogue, need for rapid test suitable for under-resourced laboratories catering point-of-care (PoC) services was construed. Present study deals with development of rapid sheep (Ovis aries) meat identification technique using DNA extraction by alkaline lysis (AL) and loop-mediated isothermal amplification (LAMP) technique. The AL-LAMP specifically amplifies sheep-specific signal of mitochondrial D loop region under an isothermal temperature of 60°C with an analytical sensitivity of 0.5ng sheep DNA. The test was highly specific to sheep and performed well even in the presence of DNA of closely related meat animal species such as goat, cattle, buffalo and chicken. The novel primers designed for the AL-LAMP successfully detected sheep meat in raw and cooked meat samples heated up to 121°C for 30min. Sheep-specific AL-LAMP assay could detect 0.1% mutton-in-beef adulteration. Novel AL-LAMP assay being simple, rapid and reliable for sheep meat authentication in just 120min; hence, it could be conveniently used by terminal laboratories engaged in rendering on-site or PoC services.

A loop-mediated isothermal amplification (LAMP) assay for detection of Toxoplasma gondii infection in women with spontaneous abortion.

The present study aimed to use the loop-mediated isothermal amplification (LAMP) technique in comparison with serological tests to determine the rate ofT. gondii infection in women suffering from spontaneous abortion (SA). A total of 140 women suffering from their first SA were included in this study. The collected aborted fetal remains and blood samples from each patient were examined in sterilized conditions using the LAMP technique and ELISA. Of the 140 women, 80 (57.1%) tested seropositive for anti-Toxoplasma antibodies by ELISA, 72 (51.4%) women tested seropositive for the IgG antibody, 8 (5.7%) tested seropositive for the IgM antibody. Among the eight women who'd had their first SA who tested seropositive for IgM antibody by ELISA, only five cases (62.5%) reported positively to the LAMP test. The difference in the frequency distribution of the LAMP results for measuring the Toxoplasma infection in pregnant women under study was statistically significant (P < 0.001) from the results of the serological test (ELISA). Although there was a significant difference between age and positivity in the LAMP test (P = 0.017), no significant difference was observed between positivity in the LAMP test and other variables. The findings of the present investigation suggest that LAMP is a preferred method for determining Toxoplasma infection in pregnant women suffering from SA compared with other routine serological tests. Even in a field with limited facilities and equipment, this technique can be effective and efficient in accurately and specifically diagnosing Toxoplasma infections in women at high risk of SA.

Multiplex Recombinase Polymerase Amplification Assay for Simultaneous Detection of Treponema pallidum and Haemophilus ducreyi in Yaws-Like Lesions.

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 min at 42 °C. The RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Comparing the developed assay with commercial multiplex real-time PCR, the assay demonstrated 94% and 95% sensitivity for T. pallidum and H. ducreyi, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication efforts by ensuring reliable diagnosis, to enable monitoring of program success and planning of follow-up interventions at the community level.

The Recent Development of Hybridization Chain Reaction Strategies in Biosensors

With the continuous development of biosensors, researchers have focused increasing attention on various signal amplification strategies to pursue superior performance for more applications. In comparison with other signal amplification strategies, hybridization chain reaction (HCR) as a powerful signal amplification technique shows its certain charm owing to nonenzymatic and isothermal features. Recently, on the basis of conventional HCR, this technique has been developed and improved rapidly, and a variety of HCR-based biosensors with excellent performance have been reported. Herein, we present a systematic and critical review on the research progress of HCR in biosensors in the last five years, including the newly developed HCR strategies such as multibranched HCR, migration HCR, localized HCR, in situ HCR, netlike HCR, and so on, as well as the combination strategies of HCR with isothermal signal amplification techniques, nanomaterials, and functional DNA molecules. By illustrating some representative works, we also summarize the advantage and challenge of HCR in biosensors, and offer a deep discussion of the latest progress and future development trends of HCR in biosensors.

Bioprocessing and integration of a high flux screening systematic platform based on isothermal amplification for the detection on 8 common pathogens

During a large variety of common pathogens, E. coli, P. aeruginosa, MRSA, MRCNS, V. parahaemolyticus, L. monocytogenes and Salmonella are the leading pathogens responsible for large number of human infections and diseases. In this study, a high flux screening based on nucleic acid isothermal amplification technique has been developed. For the 8 common pathogens, species-specific targets had been selected and analyzed for their unique specificity. After optimization, separate LAMP reaction assays had been bioprocessed and integrated into one systematic detection platform, including 8 strips (PCR tubes) and 96-well plates. Eight standard strains verified for the accuracy. Application of the established high flux screening platform was used for detection for 48 samples in 4 different 96-well plates, with 2 groups of 2 operators using double-blind procedure. The accuracy of 100% was obtained, with the total time consumption as 66–75 min (for 12 samples detection on 8 different pathogens). As concluded, through the bioprocess of the systematic platform based on LAMP technique, it’s been demonstrated to be capable of simultaneous detection of 8 pathogens, with high sensitivity, specificity, rapidity and convenience.