Isothermal Amplification Detection Research Articles

Preliminary screening method of Escherichia coli in oral drugs by visual LAMP technology

According to Chinese Pharmacopoeia, Escherichia coli should not be detected in oral drugs. In this study, a visual LAMP method for rapid detection of Escherichia coli was developed to replace the traditional microbial limit test method. Methods Four specific primers (FIP, BiP, F3, B3) for loop mediated isothermal amplification were designed by using uidA gene of Escherichia coli, and calcein-Mn2+ dye was added to the system to establish a visual loop mediated isothermal amplification detection method for Escherichia coli Finally, the visual LAMP system was used to detect the presence of E.coli and the sensitivity of the system in drugs. Results The specificity of the optimized system was good, using this system to detect 10 different strains, the results showed that only Escherichia coli genome appeared, the system turned green, the other systems were brown yellow; the detection limit of gradient dilution Escherichia coli was 102 CFU/mL by LAMP. Visual LAMP was used to detect the oral drugs sold in the market and not passed the test of the drug administration, and no E.coli was detected, but the oral drugs that were artificially contaminated with E.coli were visualized by LAMP. The color detection limit of the system in oral drugs was 1.8×102 CFU/mL. Conclusion The visual LAMP detection system can be used for the preliminary detection of oral drugs containing Escherichia coli.

Development and Evaluation of the Rapid and Sensitive RPA Assays for Specific Detection of Salmonella spp. in Food Samples.

Salmonella spp. is among the main foodborne pathogens which cause serious foodborne diseases. An isothermal real-time recombinase polymerase amplification (RPA) and lateral flow strip detection (LFS RPA) were used to detect Salmonella spp. targeting the conserved sequence of invasion protein A (invA). The Real-time RPA was performed in a portable florescence scanner at 39°C for 20 min. The LFS RPA was performed in an incubator block at 39°C for 15 min, under the same condition that the amplifications could be inspected by the naked eyes on the LFS within 5 min. The detection limit of Salmonella spp. DNA using real-time RPA was 1.1 × 101 fg, which was the same with real-time PCR but 10 times higher than that of LFS RPA assay. Moreover, the practicality of discovering Salmonella spp. was validated with artificially contaminated lamb, chicken, and broccoli samples. The analyzing time dropped from 60 min to proximately 5–12 min on the basis of the real-time and LFS RPA assays compared with the real-time PCR assay. Real-time and LFS RPA assays’ results were equally reliable. There was no cross-reactivity with other pathogens in both assays. In addition, the assays had good stability. All of these helped to show that the developed RPA assays were simple, rapid, sensitive, credible, and could be a potential point-of-need (PON) test required mere resources.

Home testing for COVID-19: Benefits and limitations.

The home test kits for detecting SARS-CoV-2 infection with Food and Drug Administration emergency use authorization primarily use either isothermal nucleic acid amplification or antigen detection, and each test has advantages and limitations in terms of sensitivity and specificity, cost, results reporting, and results turnaround time. In clinical studies, these tests provide accurate positive results in symptomatic individuals, although negative results are less accurate. There are also accuracy concerns for positive results in asymptomatic individuals. These factors have implications for their clinical interpretation and use.

Development of a point-of-care test to detect SARS-CoV-2 from saliva which combines a simple RNA extraction method with colorimetric reverse transcription loop-mediated isothermal amplification detection.

The new coronavirus infection (COVID-19) is a major public health concern, with a high burden and risk for infection among patients and healthcare workers. Saliva droplets containing SARS-COV-2 are a major vector for COVID-19 infection, making saliva a promising alternative for COVID-19 testing using nasopharyngeal swab samples. To diagnose COVID-19 patients in the field, a point-of-care test (POCT) using saliva was conceptualized. We have developed a simple method for extracting RNA from saliva samples using semi-alkaline proteinase, a sputum homogenizer typically used for preparing samples for tuberculosis testing, and a subsequent simple heating step with no need for centrifugation or RNA extraction. Further, we newly developed a triplex reverse transcription loop-mediated isothermal amplification approach (RT-LAMP) which utilizes colorimetric readout using a heat block, with results evaluated with the unaided eye. In 44 clinical patients suspected of having COVID-19 infection, the test took 45 min, and resulted in a diagnostic sensitivity of 82.6% (19/23) and diagnostic specificity of 100% (21/21), compared to the reference standard. The limit of detection was 250 copies/reaction (25,000 copies/mL). Our newly developed POCT approach achieved simple RNA extraction and constant RT-LAMP detection. This POCT has the potential to be used for simple inspection stations in a field setting, helping reduce the risk of infection by simplifying and accelerating testing for COVID-19.

First Report on the Rapid Detection and Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) in Viable but Non-culturable (VBNC) Under Food Storage Conditions.

Formation of viable but non-culturable (VBNC) status in methicillin-resistant Staphylococcus aureus (MRSA) has never been reported, and it poses a significant concern for food safety. Thus, this study aimed to firstly develop a rapid, cost-effective, and efficient testing method to detect and differentiate MRSA strains in the VBNC state and further apply this in real food samples. Two targets were selected for detection of MRSA and toxin, and rapid isothermal amplification detection assays were developed based on cross-priming amplification methodology. VBNC formation was performed for MRSA strain in both pure culture and in artificially contaminated samples, then propidium monoazide (PMA) treatment was further conducted. Development, optimization, and evaluation of PMA-crossing priming amplification (CPA) were further performed on detection of MRSA in the VBNC state. Finally, application of PMA-CPA was further applied for detection on MRSA in the VBNC state in contaminated food samples. As concluded in this study, formation of the VBNC state in MRSA strains has been verified, then two PMA-CPA assays have been developed and applied to detect MRSA in the VBNC state from pure culture and food samples.

Lab in a tube: Isolation, extraction, and isothermal amplification detection of exosomal long noncoding RNA of gastric cancer

Tumor-derived exosomes that inherit molecular information on parental cells hold great promise for cancer diagnostics. Currently, two main technical challenges, time-consuming and labor-intensive isolation of exosome and nucleic acid extraction with limited recovery that have restricted the detection of ultralow abundance exosomal nucleic acids. Here, we proposed a simple, efficient and “lab in a tube” system for the detection of exosomal nucleic acids, which fully integrated exosomes enrichment using immunomagnetic beads (IMB) (10 min), fast exosomes lysis based on NP-40 lysate (5 min) and sensitive loop-mediated isothermal amplification (LAMP) in a tube. This method was demonstrated by detecting two exosomal long noncoding RNA biomarkers of gastric cancer (HOTTIP and lncRNA-GC1) with a dynamic detection ranging from 300 ng/μL to 10 ng/μL, and the detection limit of LAMP was 10 ng/μL. Additionally, this platform exhibited good performance in the analysis of exosomal HOTTIP RNA directly in human serum samples, which has the potential for detection of low-abundance exosomal nucleic acid biomarkers from cancers.

All-in-one in situ colorimetric RT-LAMP assay for point-of-care testing of SARS-CoV-2.

The ongoing outbreaks of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have resulted in unprecedented challenges to global health. To effectively contain the COVID-19 transmission, rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. To address the huge need for ever-increasing tests, we developed a facile all-in-one nucleic acid testing assay by combining Si-OH activated glass bead (aGB)-based viral RNA fast extraction and in situ colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection in a single tube. aGBs demonstrate a strong ability to capture viral RNA in a guanidinium-based lysis buffer, and the purified aGBs/RNA composite, without RNA elution step, could be directly used to perform RT-LAMP assay. The assay was well characterized by using a novel SARS-CoV-2-like coronavirus GX/P2V, and showed a limit of detection (LOD) of 15 copies per μL in simulated clinical samples within 50 min. We further demonstrated our assay by testing simulated SARS-CoV-2 pseudovirus samples, showing an LOD of 32 copies per μL and high specificity without cross-reactivity with the most closely related GX/P2V or host DNA/RNA. The all-in-one approach developed in this study has the potential as a simple, scalable, and time-saving alternative for point-of-care testing of SARS-CoV-2 in low-income regions, as well as a promising tool for at-home testing.

An integrated microfluidic detection system for the automated and rapid diagnosis of high-risk human papillomavirus.

Human papillomavirus (HPV) causes the prevalent sexually transmitted infection that accounts for the majority of cervical cancer incidences. Therefore, the development of a rapid, accurate, automatic and affordable nucleic acid detection strategy is urgently required for HPV tests, among which microfluidic chip is a promising diagnostic method. In this work, we developed a microfluidic detection system consisting of a microfluidic chip and the corresponding detection equipment to diagnose high-risk HPV. The proposed method integrates nucleic acid purification, isothermal amplification and real-time fluorescence detection into one device. Moreover, it demonstrates good detection performance such as high specificity of primer sets (100%) and exceptional stability (coefficient of variation <6%) among five HPV genotypes. Besides, the microfluidic loop-mediated isothermal amplification (LAMP) assay is accurate (specificity of 91.7% and sensitivity of 100%) and fast (average time threshold = 10.56 minutes) when considering the conventional qPCR assay as the gold standard. The integrated microfluidic detection system offers automated and rapid diagnosis within 40 minutes and shows broad potential to deliver point-of-care detection in resource-limited circumstances owing to its simplicity and affordability.

Rapid Loop-Mediated Isothermal Amplification for Detection of the Ralstonia solanacearum Species Complex Bacteria in Symptomatic Potato Tubers and Plants.

The Ralstonia solanacearum species complex (RSSC) is composed of several Ralstonia species and strains that are little related and show varied host range and distinct geographic distributions. The RSSC causes wilt disease, and can thus have severe economic consequences for many important crops and ornamental plants. One such is potato (Solanum tuberosum), where infection causes brown rot of the tubers. It is important that symptomatic tubers and plants can be rapidly and easily tested, as exclusion of infected material is a cornerstone of management of bacterial diseases. A suitable method is loop-mediated isothermal amplification, a rapid, DNA-based method that can be used for specific detection of plant pathogens in infected materials. The combination of this loop-mediated isothermal amplification assay for the RSSC with a simple sample preparation method is fit for purpose for identification of this devastating disease in symptomatic tubers and plants. This methodology is rapid and cost efficient, and can be carried out outside of conventional laboratory facilities.

Bioluminescent detection of isothermal DNA amplification in microfluidic generated droplets and artificial cells

Microfluidic droplet generation affords precise, low volume, high throughput opportunities for molecular diagnostics. Isothermal DNA amplification with bioluminescent detection is a fast, low-cost, highly specific molecular diagnostic technique that is triggerable by temperature. Combining loop-mediated isothermal nucleic acid amplification (LAMP) and bioluminescent assay in real time (BART), with droplet microfluidics, should enable high-throughput, low copy, sequence-specific DNA detection by simple light emission. Stable, uniform LAMP–BART droplets are generated with low cost equipment. The composition and scale of these droplets are controllable and the bioluminescent output during DNA amplification can be imaged and quantified. Furthermore these droplets are readily incorporated into encapsulated droplet interface bilayers (eDIBs), or artificial cells, and the bioluminescence tracked in real time for accurate quantification off chip. Microfluidic LAMP–BART droplets with high stability and uniformity of scale coupled with high throughput and low cost generation are suited to digital DNA quantification at low template concentrations and volumes, where multiple measurement partitions are required. The triggerable reaction in the core of eDIBs can be used to study the interrelationship of the droplets with the environment and also used for more complex chemical processing via a self-contained network of droplets, paving the way for smart soft-matter diagnostics.

Development and validation of Loop-Mediated Isothermal Amplification (LAMP) detection limit using blood matrix spike for Candida glabrata detection

Background: Rapid and accurate identification of yeast responsible for invasive candidiasis (IC) allows for focused and early initiation of therapy for good clinical outcome. Candida glabrata is increasingly important, and the diagnostic sensitivity of microbial culture method “gold standard” is low and could miss up to 50% of the IC patients. To develop and validate LAMP technique detection limit in comparison to microbial culture and PCR techniques using blood matrix spike for C. glabrata detection. Methods and materials: Blood spiking experiment was conducted using healthy blood sample. Candida glabrata ATCC 2001 reference strain colonies were re-suspended in phosphate-buffered saline (PBS) buffer to 0.5 McFarland standard. Ten-fold serial dilutions of yeast suspensions in 1x PBS was made from 106 to 101 cfu/mL concentrations. A 100 μL from each dilution was plated onto SDA and the resulting number of colonies was established. A 25 μL of each dilution was spiked in 225 μL of blood sample (1:10 dilution). The C. glabrata DNA was extracted from spiked blood using Qiagen yeast DNA extraction kit. The developed LAMP assay detects C. glabrata using a primer set (three pairs) (FIP/BIP, F3/B3 and LF/LB) that recognized eight distinct regions on the target internal transcribed spacer (ITS) gene, while, ITS1/4 universal primers were used in PCR analysis. The cfu/mL detection limit of the LAMP was compared to culture and PCR techniques. Results: The LAMP assay analysis of DNA from spiked blood, indicates detection limit of 101 cfu/mL (4.03 x 101 cells/25 μL) using gel electrophoresis analysis. The PCR detection limit was 105 cfu/mL (4.03 x 104 cells/25 μL) and the culture detection limit was 104 cfu/mL (1.61 x 106 cells/mL). Conclusion: The LAMP assay indicates high sensitivity of C. glabrata ITS detection from spiked blood compared to PCR and therefore, a good diagnostic tool for molecular analysis.

Suitcase Lab: new, portable, and deployable equipment for rapid detection of specific harmful algae in Chilean coastal waters

Phytoplankton blooms, including harmful algal blooms (HABs), have serious impacts on ecosystems, public health, and productivity activities. Rapid detection and monitoring of marine microalgae are important in predicting and managing HABs. We developed a toolkit, the Suitcase Lab, to detect harmful algae species in the field. We demonstrated the Suitcase Lab’s capabilities for sampling, filtration, DNA extraction, and loop-mediated isothermal amplification (LAMP) detection in cultured Alexandrium catenella cells as well as Chilean coastal waters from four sites: Repollal, Isla García, Puerto Montt, and Metri. A LAMP assay using the Suitcase Lab in the field confirmed microscopic observations of A. catenella in samples from Repollal and Isla García. The Suitcase Lab allowed the rapid detection of A. catenella, within 2 h from the time of sampling, even at a single cell per milliliter concentrations, demonstrating its usefulness for quick and qualitative on-site diagnosis of target toxic algae species. This method is applicable not only to detecting harmful algae but also to other field studies that seek a rapid molecular diagnostic test.

A diagnostic test that uses isothermal amplification and lateral flow detection sdaA can detect tuberculosis in 60min.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is now the leading cause of death from infectious disease, thus rapid diagnostic and screening techniques for TB are urgently needed. Here, a detection of MTB using multiple cross displacement amplification coupling with nanoparticles-based lateral flow device (MCDA-LFD) was developed and validated, targeting the specific sdaA gene. The whole detection procedure, including rapid genomic DNA extraction (15min), amplification (30min) and result reporting (2min), was completed within 50min. No cross-reaction with non-mycobacteria and non-tuberculous mycobacteria (NTM) strains was observed. The sensitivity of sdaA-MCDA-LFD, Xpert MTB/RIF assay and culture results was 81·6, 48·3 and 37·9%, respectively, in TB patients. Among positive culture samples, the sensitivity of sdaA-MCDA-LFD and Xpert MTB/RIF assay was 93·9% (31/33) and 81·8% (27/33), respectively. Among culture-negative samples, the sensitivity of sdaA-MCDA-LFD and Xpert MTB/RIF assay was 74·1% (40/54) and 27·8% (15/54), respectively. The specificity of sdaA-MCDA-LFD and Xpert MTB/RIF was 95·4% (62/65) and 100% (65/65) in clinical samples from non-TB patients. The sdaA-MCDA-LFD assay was a rapid, simple, specific and sensitive TB diagnostic test. The sdaA-MCDA-LFD assay holds promise for application as a useful point-of-care test to detect MTB, and will play an important role in controlling and preventing TB.

Cleavable hairpin beacon-enhanced fluorescence detection of nucleic acid isothermal amplification and smartphone-based readout

Fluorescence detection of nucleic acid isothermal amplification utilizing energy-transfer-tagged oligonucleotide probes provides a highly sensitive and specific method for pathogen detection. However, currently available probes suffer from relatively weak fluorescence signals and are not suitable for simple, affordable smartphone-based detection at the point of care. Here, we present a cleavable hairpin beacon (CHB)-enhanced fluorescence detection for isothermal amplification assay. The CHB probe is a single fluorophore-tagged hairpin oligonucleotide with five continuous ribonucleotides which can be cleaved by the ribonuclease to specifically initiate DNA amplification and generate strong fluorescence signals. By coupling with loop-mediated isothermal amplification (LAMP), the CHB probe could detect Borrelia burgdorferi (B. burgdorferi) recA gene with a sensitivity of 100 copies within 25 min and generated stronger specific fluorescence signals which were easily read and analysed by our programmed smartphone. Also, this CHB-enhanced LAMP (CHB-LAMP) assay was successfully demonstrated to detect B. burgdorferi DNA extracted from tick species, showing comparable results to real-time PCR assay. In addition, our CHB probe was compatible with other isothermal amplifications, such as isothermal multiple-self-matching-initiated amplification (IMSA). Therefore, CHB-enhanced fluorescence detection is anticipated to facilitate the development of simple, sensitive smartphone-based point-of-care pathogen diagnostics in resource-limited settings.

Development of Sars-Cov-2 Isothermal Amplification Detection Kits

The SARS-CoV-2 isothermal amplification detection kits based on loop-mediated isothermal amplification (LAMP) were developed and evaluated on three types samples of SARS-CoV-2 The kits included enzyme reaction mixtures and chromogenic agents After the isothermal amplification reactions were completed, the reaction results were judged by using the chromogenic agents to determine whether SARS-CoV-2 exists in the samples to be tested The detection kits have the advantages of convenient operation, fast detection speed and high sensitivity up to 1 copy of virus particles per reaction, which can speed up the detection speed of suspected cases, and avoid the missing detection problems caused by the low detection sensitivity

Detection of Salmonella by the 3M Molecular Detection Assays: MDS® Method.

Developed by 3M Company, 3M ™ Molecular Detection Assays-3M MDS-enable detection of Salmonella from advanced isothermal DNA amplification and bioluminescence detection technology. It can be used for a wide variety of products, including poultry, eggs, pet foods, and environmental samples, and results are obtained within about 24h. In this chapter, all steps of the 3M MDS™ method for detection of Salmonella are described and detailed.

Rapid isothermal amplification and portable detection system for SARS-CoV-2.

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

Rapid and semi-quantitative colorimetric loop-mediated isothermal amplification detection of ASFV via HSV color model transformation.

African Swine Fever (ASF) is a highly contagious and lethal viral disease of swine, the presence of which in groups of pigs leads to enormous economic losses in the farming industry. However, vaccines and drugs to treat ASF have yet to be developed. To control the spread of the African Swine Fever Virus (ASFV), a diagnostic method that can be applied rapidly and can detect the disease during the early stages of infection is urgently needed. In this study, we demonstrate a rapid and easy-to-use ASFV detection method that combines loop-mediated isothermal amplification (LAMP) and image processing with the hue-saturation-value (HSV) color model. This method was validated through use of synthetic ASFV DNA. The method shows high sensitivity, as it detects as few as 10 copies per reaction within 20min. The speed and sensitivity of this newly developed assay are superior to those reported in previous studies. In addition, through HSV color space transformation, the colorimetric result of this LAMP assay can be used for a semi-quantitative analysis for ASFV (ranging from 108 to 101 copies per reaction) and improve the discern to low concentration samples from a negative control. These results show that the combination of ASFV-LAMP assay and HSV color space transformation may accelerate the screening process of pigs for ASFV infection. Overall, this study provides a rapid, sensitive, early-stage, on-site diagnosis of ASFV infection and has potential to be applied to other infectious diseases.

Genotyping of 30 kinds of cutaneous human papillomaviruses by a multiplex microfluidic loop-mediated isothermal amplification and visual detection method

BackgroundHuman papillomaviruses (HPVs), a group of non-enveloped small viruses with double-stranded circular DNA which lead to multiple skin diseases such as benign warts, are commonly seen in clinics. The current HPV detection systems aim mainly at mucosal HPVs, however, an efficient clinical approach for cutaneous HPVs detection is lacking.ObjectivesTo establish a rapid detection system for cutaneous HPVs using a colorimetric loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue (HNB) dye in combination with microfluidic technology.MethodsL1 DNA sequences of the 30 cutaneous HPVs were chemically synthesized, and LAMP primers against L1 DNA were designed with use of an online LAMP designing tool. Isothermal amplification was performed with use of a water bath and the amplification results were inspected with the naked eye. Using PCR sequencing as a control method, the specificity and sensitivity of the new detection system were obtained by detecting clinical samples.ResultsThe lower detection limit of the LAMP assay was 107 viral DNA copies/μl when tested on synthesized L1 DNA sequences, which was better than the conventional PCR. Compared to PCR sequencing, the sensitivity of HPV27, HPV2, HPV1, HPV57, HPV3, HPV4, HPV7 and HPV75 genotypes detections were 100%, whereas the specificity was 34.55, 45.12, 95.83, 98.59 and 97.62% respectively, when tested on clinical samples.ConclusionsThe new cutaneous type HPV detection system is characterized by both a good sensitivity and specificity compared to conventional methods.

Multiple-centre clinical evaluation of an ultrafast single-tube assay for SARS-CoV-2 RNA

ObjectiveTo evaluate the performance of an ultrafast single-tube nucleic acid isothermal amplification detection assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA using clinical samples from multiple centres. MethodsA reverse transcription recombinase–aided amplification (RT-RAA) assay for SARS-CoV-2 was conducted within 15 minutes at 39°C with portable instruments after addition of extracted RNA. The clinical performance of RT-RAA assay was evaluated using 947 clinical samples from five institutions in four regions of China; approved commercial fluorescence quantitative real-time PCR (qRT-PCR) kits were used for parallel detection. The sensitivity and specificity of RT-RAA were compared and analysed. ResultsThe RT-RAA test results of 926 samples were consistent with those of qRT-PCR (330 were positive, 596 negative); 21 results were inconsistent. The sensitivity and specificity of RT-RAA was 97.63% (330/338, 95% confidence interval (CI) 95.21 to 98.90) and 97.87% (596/609, 95% CI 96.28 to 98.81) respectively. The positive and negative predictive values were 96.21% (330/343, 95% CI 93.45 to 97.88) and 98.68% (596/604, 95% CI 97.30 to 99.38) respectively. The total coincidence rate was 97.78% (926/947, 95% CI 96.80 to 98.70), and the kappa was 0.952 (p < 0.05). ConclusionsWith comparable sensitivity and specificity to the commercial qRT-PCR kits, RT-RAA assay for SARS-CoV-2 exhibited the distinctive advantages of simplicity and rapidity in terms of operation and turnaround time.