Loop-mediated Amplification Research Articles

Benchtop to at-home test: Amplicon-depleted CRISPR-regulated loop mediated amplification at skin-temperature for viral load monitoring

CoRPLA (CRISPR-regulated One-pot Recombinase Polymerase Loop-mediated Amplification) is an amplicon-depleted skin-temperature operated iNAAT designed for at-home testing. It uses specially designed loop primers to enhance isothermal amplification, triggering Cas12 for in-situ amplicon depletion and signal amplification. This method addresses issues like amplicon-derived aerosol contamination and complex assay formats, enabling quantitative detection with sub-attomolar sensitivity (0.5 cps/μL). CoRPLA employs a DNA hydrogel wearable tape for real-time, colorimetric readout, allowing visual differentiation of pathogen loads. It was validated with clinical samples for SARS-CoV-2, RSV, influenza A, and HPV, successfully identifying multi-level viral loads of the positive cases with results consistent with qPCR. Offering high sensitivity while eliminating false positives from aerosol contamination, CoRPLA bridges the molecular assay from benchtop to home for daily viral infections monitoring.

LAMP-based electrochemical platform for monitoring HPV genome integration at the mRNA level associated with higher risk of cervical cancer progression.

Human papillomaviruses (HPVs) represent a diverse group of double-stranded DNA viruses associated with various types of cancers, notably cervical cancer. High-risk types of HPVs exhibit their oncogenic potential through the integration of their DNA into the host genome. This integration event contributes significantly to genomic instability and the progression of malignancy. However, traditional detection methods, such as immunohistochemistry or PCR-based assays, face inherent challenges, and thus alternative tools are being developed to fasten and simplify the analysis. Our study introduces an innovative biosensing platform that combines loop-mediated amplification with electrochemical (EC) analysis for the specific detection of HPV16 integration. By targeting key elements like the E7 mRNA, a central player in HPV integration, and the E2 viral gene transcript lost upon integration, we show clear distinction between episomal and integrated forms of HPV16. Our EC data confirmed higher E7 expression in HPV16-positive cell lines having integrated forms of viral genome, while E2 expression was diminished in cells with fully integrated genomes. Moreover, we revealed distinct expression patterns in cervical tissue of patients, correlating well with digital droplet PCR, qRT-PCR, or immunohistochemical staining. Our platform thus offers insights into HPV integration in clinical samples and facilitates further advancements in cervical cancer research and diagnostics.

Evaluation of the Eazyplex®Candida ID LAMP Assay for the Rapid Diagnosis of Positive Blood Cultures.

Rapid molecular assays can be used to identify Candida pathogens directly from positive blood cultures (BCs) in a timely manner compared to standard methods using subcultures. In this study, the eazyplex®Candida ID assay, which is based on loop-mediated amplification (LAMP) and is currently for research use only, was evaluated for the identification of the most common fungal species. A total of 190 BCs were analysed. Sensitivity and specificity were 93.88% and 99.26% for C. albicans, 89.13% and 100% for Nakaseomyces glabratus (N. glabratus), 100% and 100% for Pichia kudravzevii (P. kudriavzevii), 100% and 100% for C. tropicalis, and 100% and 99.44% for C. parapsilosis. Sample preparation took approximately 11 min and positive amplification results were obtained between 8.5 and 19 min. The eazyplex®Candida ID LAMP assay is an easy-to-use diagnostic tool that can optimise the management of patients with candidemia.

Rapid Detection of Viral, Bacterial, Fungal, and Oomycete Pathogens on Tomatoes with Microneedles, LAMP on a Microfluidic Chip, and Smartphone Device.

Rapid detection of plant diseases before they escalate can improve disease control. Our team has developed rapid nucleic acid extraction methods with microneedles and combined these with loop-mediated amplification (LAMP) assays for pathogen detection in the field. In this work, we developed LAMP assays for early blight (Alternaria linariae, A. alternata, and A. solani) and bacterial spot of tomato (Xanthomonas perforans) and validated these LAMP assays and two previously developed LAMP assays for tomato spotted wilt virus and late blight. Tomato plants were inoculated, and disease severity was measured. Extractions were performed using microneedles, and LAMP assays were run in tubes (with hydroxynaphthol blue) on a heat block or on a newly designed microfluidic slide chip on a heat block or a slide heater. Fluorescence on the microfluidic chip slides was visualized using EvaGreen and photographed on a smartphone. Plants inoculated with X. perforans or tomato spotted wilt virus tested positive prior to visible disease symptoms, whereas Phytophthora infestans and A. linariae were detected at the time of visual disease symptoms. LAMP assays were more sensitive than PCR, and the limit of detection was 1 pg of DNA for both A. linariae and X. perforans. The LAMP assay designed for early blight detected all three species of Alternaria that infect tomato and is thus an Alternaria spp. assay. This study demonstrates the utility of rapid microneedle extraction followed by LAMP on a microfluidic chip for rapid diagnosis of four important tomato pathogens.

Rapid detection of grapevine berry inner necrosis virus using a simple reverse transcription loop-mediated amplification method

To develop a simple method for the detection of grapevine berry inner necrosis (GINV) that does not depend on expensive instruments, a reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay was developed. A set of primers was used in RT-LAMP to specifically detect GINV and different GINV variants belonging to groups 1, 2 and 3. The sensitivity of the RT-LAMP was about 100 times greater than that of conventional RT-PCR. Furthermore, a rapid “pin-prick” assay without RNA extraction was developed to detect GINV in RT-LAMP. The assay performed well for the detection of GINV from different parts of field grapevine plants and micropropagated grapevine plantlets, and had a higher detection rate of GINV than RT-PCR in the tested grapevine samples.

Next step towards point-of-care molecular diagnosis of female genital schistosomiasis (FGS): evaluation of an instrument-free LAMP procedure

Detection of Schistosoma spp. DNA in gynaecological samples by quantitative real-time polymerase chain reaction (qPCR) is considered to be the reference diagnostic test for female genital schistosomiasis (FGS). However, qPCR needs expensive laboratory procedures and highly trained technicians. Loop-mediated amplification (LAMP) is a more field-friendly isothermal procedure for the detection of parasite-specific DNA, but it still requires electrically powered equipment. Here, we validated a Schistosoma haematobium-specific Sh-LAMP procedure and tested a fully instrument-free isothermal amplification using a novel low-cost, and reusable Temperature-cup (T-cup) device. Specific primers were selected based on published assays, targeting the ribosomal intergenic spacer (IGS) region of S. haematobium. Technical validation of the IGS-Sh-LAMP was performed using 20 negative controls, including DNA extracts of soil-transmitted helminths and S. mansoni, and a 10-fold dilution series (100–10−3) of DNA extracted from a single S. haematobium egg (n=4). For clinical validation, the IGS-Sh-LAMP was tested on 125 DNA samples extracted from vaginal swabs of a previous FGS study in Madagascar. Results were compared with the quantification cycle value (Cq) of the standard ITS-2 targeting qPCR. Single S. haematobium egg DNA up to a 10–2 dilution and an ITS-2 Cq <35 tested positive in the IGS-Sh-LAMP. The specificity was found to be excellent (100%). In the clinical samples, IGS-Sh-LAMP showed comparable results with the qPCR, with 35.2% and 33.6% positives, respectively, and a concordance of 79.2% (99/125). Of the 12 false-negatives, 5 corresponded to the 7 qPCR positive samples with very low DNA levels (Cq ≥35). On the other hand, IGS-Sh-LAMP detected 14 additional cases that were not detected by qPCR. The T-cup IGS-Sh-LAMP performance was evaluated in a representative sub-selection (n=10) of IGS-Sh-LAMP positive clinical samples. The T-cup IGS-Sh-LAMP was found to be a very user-friendly method, but in different runs, it missed 1 to 4 of the 10 IGS-Sh-LAMP positive samples, specifically those with a low DNA load. Our results show that the IGS-Sh-LAMP is a suitable alternative to the ITS-2 qPCR for the diagnosis of FGS in gynaecological samples, with high potential for the T-cup as a fully instrument-free isothermal amplification device for point-of-care diagnosis in low-resource settings.

A Diagnostic Chip for the Colorimetric Detection of Legionella pneumophila in Less than 3 h at the Point of Need.

Legionella pneumophila has been pinpointed by the World Health Organization as the highest health burden of all waterborne pathogens in the European Union and is responsible for many disease outbreaks around the globe. Today, standard analysis methods (based on bacteria culturing onto agar plates) need several days (~12) in specialized analytical laboratories to yield results, not allowing for timely actions to prevent outbreaks. Over the last decades, great efforts have been made to develop more efficient waterborne pathogen diagnostics and faster analysis methods, requiring further advancement of microfluidics and sensors for simple, rapid, accurate, inexpensive, real-time, and on-site methods. Herein, a lab-on-a-chip device integrating sample preparation by accommodating bacteria capture, lysis, and DNA isothermal amplification with fast (less than 3 h) and highly sensitive, colorimetric end-point detection of L. pneumophila in water samples is presented, for use at the point of need. The method is based on the selective capture of viable bacteria on on-chip-immobilized and -lyophilized antibodies, lysis, the loop-mediated amplification (LAMP) of DNA, and end-point detection by a color change, observable by the naked eye and semiquantified by computational image analysis. Competitive advantages are demonstrated, such as low reagent consumption, portability and disposability, color change, storage at RT, and compliance with current legislation.

DEVELOPMENT AND VALIDATION OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) FOR DETECTION OF LEPTOSPIR GENETIC MATERIAL

Primer systems were designed and tested and the method of loop-mediated isothermal amplification was validated for rapid diagnosis of leptospirosis. In general, five sets of primers were calculated and analyzed in situ and in vitro, which were ordered for synthesis and chosen for the future as the most promising. Indicators of analytical sensitivity and detection limits of the method were established, the specificity of the method in its tests with homologous and close to taxonomic characteristics heterologous samples of genetic material was proved and completed reproducibility of the method was established under the conditions of its staging on various laboratory equipment and with reagents from different manufacturers. The developed technique of loop-mediated amplification for the diagnosis of leptospirosis was tested on field samples and its diagnostic effectiveness was established.

Review on Current Diagnostic Methods of Bovine Mastitis

Mastitis is an inflammation of the mammalian gland, and it is a commercially significant ailment in the dairy industry worldwide. Based on the source, it can be either contagious or environmental; on the other hand, clinical and subclinical mastitis are based on their severity. The most important step between disease etiology and cure is accurate disease diagnosis. A cost-effective, dependable, and quick diagnostic instrument is essential for udder health management. Therefore, the objective of this article is to review current diagnostic methods for bovine mastitis. The diagnostic methods include clinical diagnosis, microscopically examined specimens, conventional methods, power of hydrogen (pH) tests, microbial culture, molecular methods, and those based on emerging technology. Conventional methods like the strip cup test, California mastitis test, Wisconsin mastitis test, somatic cell count test, and electrical conductivity test are used as screening tests that are not specific to any pathogen. Microbial culture-based methods are the gold standard for identifying mastitis pathogens and are frequently used, but they have some drawbacks. Advanced molecular methods such as polymerase chain reaction and its version, loop-mediated amplification, are used for the identification of causative agents at the strain level. Emerging technologies such as infrared thermography and biosensors have the potential to match or surpass the diagnostic methods mentioned above concerning sensitivity, selectivity, accuracy, and realism. Emerging technologies are growing at a fast pace to diagnose human cases, but applications for the veterinary market, and specifically, for the diagnosis of mastitis, remain limited. Both specializations in training and experience in the interpretation of results are needed for implementing advanced technologies for efficient mastitis diagnosis and improving udder health management. It should be better to adopt and employ advanced diagnostic methods, such as advanced molecular tests and emerging technology.

Real-world evaluation of the Lucira Check-It COVID-19 loop-mediated amplification (LAMP) test

ABSTRACT In hospitals during the COVID-19 pandemic, laboratory testing was important to reduce SARS-CoV-2 transmissions, particularly for high-risk settings like the emergency department and pre-operative settings and for the safe return to work of exposed healthcare workers (HCWs). For these applications, delayed test results from laboratory nucleic acid amplification tests (NAATs) posed a barrier to maximizing efficient patient flow and minimizing staffing shortages. This quality improvement project sought to evaluate the performance of the Lucira Check-It COVID-19 Test, a rapid diagnostic test that used NAAT technology (NAAT-RDT). Using 10-fold serial dilutions of SARS-CoV-2, the analytical sensitivity of the NAAT-RDT was assessed against standard NAATs used for routine diagnostic testing. Clinical performance was assessed at two Nova Scotia hospitals in 405 cases with paired swabs tested by NAAT-RDT and laboratory-based NAATs. These represented three distinct populations: patients presenting to the emergency department ( n = 208), patients in the pre-operative setting ( n = 158), and patients presenting to community testing sites ( n = 38). The analytical sensitivity of the NAAT-RDT and other laboratory NAATs was comparable. During clinical evaluation, the overall sensitivity and specificity were 92.9% and 98.3%, respectively, with little variation between settings. The Lucira NAAT-RDT is a portable and self-contained device that provides an easily interpreted result within 30 minutes following a bilateral nasal swab collection. Its performance was shown to be acceptable for use in three settings in this quality improvement project, facilitating patient flow and management. IMPORTANCE In hospitals during the COVID-19 pandemic, laboratory testing was important to reduce SARS-CoV-2 transmissions, while facilitating patient flow in the emergency department and pre-operative settings, and allowing for the safe return to work of exposed healthcare workers. Delayed test results from laboratory nucleic acid amplification tests (NAATs) posed a barrier to maximizing efficient patient flow and minimizing staffing shortages. This quality improvement project sought to evaluate the analytical and clinical performance of the Lucira Check-It COVID-19 Test, a point-of-care test that used NAAT technology, in the perioperative setting, emergency department, and community testing sites. We found the Lucira Check-It to have comparable performance to laboratory NAATs. It can be employed with little training for specimen collection, processing, and interpretation, and at a cost justifiable from the resources saved from avoiding sample transport and laboratory testing.

Diagnostic value of loop-mediated isothermal amplification in detecting lower respiratory pathogens.

To investigate the diagnostic value of loop-mediated thermostatic amplification (LAMP) in detecting pathogenic bacteria from bronchoalveolar lavage fluid (BALF) of patients with pulmonary disorders combined with lower respiratory tract infections (LRTI). This cross-sectional study included patients with pulmonary disorders combined with LRTI, including chronic obstructive pulmonary disease (COPD), bronchiectasis, or lung cancer, hospitalized in Meizhou People's Hospital between January 2020 and October 2021. BALF was collected using local bronchoalveolar lavage and electronic bronchoscopy. The presence of the pathogens was confirmed using the LAMP method and the bacterial culture method. In total, 249 patients were included (135 with COPD, 73 with bronchiectasis, and 41 with lung cancer). The proportions of Methicillin-resistant Staphylococcus aureus (4.8% vs 0.4%, p = 0.02) and Haemophilus influenzae (6.8% vs 0.4%, p < 0.001) detected by the LAMP method was higher, while the proportion of Pseudomonas aeruginosa was lower compared with that of the culture method (6.8% vs 12.4%, p = 0.034). The bacterial species with the highest agreement coefficient was Stenotrophomonas maltophilia (Kappa = 0.798, p < 0.001). Furthermore, 9 COPD patients exhibited mixed infections as determined by the LAMP method, whereas the culture method detected only 2 of these cases (1.48%) (p < 0.05). LAMP can detect more pathogenic bacteria, notably Haemophilus influenza, Methicillin-resistant Staphylococcus aureus, and atypical pathogens in patients with clinically common pulmonary disorders combined with LRTI. LAMP may provide etiological evidence to guide the clinical use of antibiotics in primary hospitals.

Unamplified and Label-Free Detection of HPV16 DNA Using CRISPR-Cas12a-Functionalized Solution-Gated Graphene Transistors.

The persistent infection of high-risk-human papillomavirus type 16 (HPV16) is considered an essential element for suffering cervical cancer. Despite polymerase chain reaction, loop-mediated amplification, and microfluidicchips are used to detect the HPV16, these methods still exist some drawbacks including time-consuming and false positive results. The CRISPR-Cas system is widely used in the region of biological detection due to its precise targeted recognition capability. In this contribution, the novel solution-gated graphene transistor sensor is designed to realize the unamplified and label-free detection of HPV16 DNA. Using the precise recognition of the CRISPR-Cas12a system and the gate functionalization, HPV16 DNA can be precisely identified without need the amplification and labeling. The limit of detection of the sensor can be up to 8.3×10-18 m and the detection can be within 20min. Additionally, the heat-Inactivated clinical samples can be clearly distinguished by the sensor the diagnosis results have a high degree of agreement with q-PCR detection.

Nanoplasmonic amplification in microfluidics enables accelerated colorimetric quantification of nucleic acid biomarkers from pathogens.

Deployment of nucleic acid amplification assays for diagnosing pathogens in point-of-care settings is a challenge due to lengthy preparatory steps. We present a molecular diagnostic platform that integrates a fabless plasmonic nano-surface into an autonomous microfluidic cartridge. The plasmonic 'hot' electron injection in confined space yields a ninefold kinetic acceleration of RNA/DNA amplification at single nucleotide resolution by one-step isothermal loop-mediated and rolling circle amplification reactions. Sequential flow actuation with nanoplasmonic accelerated microfluidic colorimetry and in conjugation with machine learning-assisted analysis (using our 'QolorEX' device) offers an automated diagnostic platform for multiplexed amplification. The versatility of QolorEX is demonstrated by detecting respiratory viruses: SARS-CoV-2 and its variants at the single nucleotide polymorphism level, H1N1 influenza A, and bacteria. For COVID-19 saliva samples, with an accuracy of 95% on par with quantitative polymerase chain reaction and a sample-to-answer time of 13 minutes, QolorEX is expected to advance the monitoring and rapid diagnosis of pathogens.

Rapid culture-independent loop-mediated isothermal amplification detection of antimicrobial resistance markers from environmental water samples.

Environmental water is considered one of the main vehicles for the transmission of antimicrobial resistance (AMR), posing an increasing threat to humans and animals health. Continuous efforts are being made to eliminate AMR; however, the detection of AMR pathogens from water samples often requires at least one culture step, which is time-consuming and can limit sensitivity. In this study, we employed comparative genomics to identify the prevalence of AMR genes within among: Escherichia coli, Klebsiella, Salmonella enterica and Acinetobacter, using publicly available genomes. The mcr-1, blaKPC (KPC-1 to KPC-4 alleles), blaOXA-48, blaOXA-23 and blaVIM (VIM-1 and VIM-2 alleles) genes are of great medical and veterinary significance, thus were selected as targets for the development of isothermal loop-mediated amplification (LAMP) detection assays. We also developed a rapid and sensitive sample preparation method for an integrated culture-independent LAMP-based detection from water samples. The developed assays successfully detected the five AMR gene markers from pond water within 1h and were 100% sensitive and specific with a detection limit of 0.0625 μg/mL and 10cfu/mL for genomic DNA and spiked bacterial cells, respectively. The integrated detection can be easily implemented in resource-limited areas to enhance One Health AMR surveillances and improve diagnostics.

New Virus Diagnostic Approaches to Ensuring the Ongoing Plant Biosecurity of Aotearoa New Zealand.

To protect New Zealand's unique ecosystems and primary industries, imported plant materials must be constantly monitored at the border for high-threat pathogens. Techniques adopted for this purpose must be robust, accurate, rapid, and sufficiently agile to respond to new and emerging threats. Polymerase chain reaction (PCR), especially real-time PCR, remains an essential diagnostic tool but it is now being complemented by high-throughput sequencing using both Oxford Nanopore and Illumina technologies, allowing unbiased screening of whole populations. The demand for and value of Point-of-Use (PoU) technologies, which allow for in situ screening, are also increasing. Isothermal PoU molecular diagnostics based on recombinase polymerase amplification (RPA) and loop-mediated amplification (LAMP) do not require expensive equipment and can reach PCR-comparable levels of sensitivity. Recent advances in PoU technologies offer opportunities for increased specificity, accuracy, and sensitivities which makes them suitable for wider utilization by frontline or border staff. National and international activities and initiatives are adopted to improve both the plant virus biosecurity infrastructure and the integration, development, and harmonization of new virus diagnostic technologies.

Rapid point of care testing for four bacterial sexually transmitted infections using the portable isothermal loop-mediated nucleic acid amplification eazyplex platform

PurposeTo analyze sensitivity and specificity of the rapid point-of-care (POC) eazyplex testing platform for bacterial sexually transmitted infections (STI) among men who have sex with men (MSM).Methods272 anal, urethral, and pharyngeal swabs collected from 153 MSM were tested by both the eazyplex platform and an in-house PCR or culture in the university microbiology reference laboratory.ResultsCompared to the reference diagnostic method, the overall sensitivity/specificity of eazyplex was 88%/98% for N. gonorrhoeae, 82%/100% for C. trachomatis, 70%/ > 99% for U. urealyticum, and 85%/98% for M. hominis, respectively. Sensitivity for N. gonorrhoeae and U. urealyticum in urethral samples was 100%.ConclusionWith good to very good sensitivity depending on the sampling site and pathogen as well as very good specificity overall the eazyplex platform is a useful rapid diagnostic method for POC bacterial STI-testing especially for N. gonorrhoeae and C. trachomatis, allowing for almost immediate treatment initiation.

A novel paper-based lysis strip for SARS-CoV-2 RNA detection at low resource settings

Infectious respiratory diseases such as COVID-19 are serious and global concerns from the past to the present. To isolate the spread of infectious diseases even in the absence of a health system, a simple, inexpensive, reliable, sensitive, and selective molecular diagnosis platform for Point of Care Test (POCT) is required. Especially, the nucleic acid extraction step is difficult to perform out of laboratory. Here, we propose a paper-based lysis (PBL) strip for nucleic acid extraction, especially in low-resource settings (LRS). PBL strips are suitable for isolating RNA from viruses with biological interference and inhibitors. We optimized the buffer compositions and membranes of the strip. A simple preparation method using a PBL strip could obtain an eluent for downstream inspection within 20 min. Overall, 104 copies/swaps were detected for 20 min for amplification in combination with Reverse Transcription Loop-Mediated Amplification (RT-LAMP).

Molecular Beacons - Loop-Mediated Amplification (MB-LAMP).

High specificity has been demonstrated in polymerase chain reaction (PCR) with the use of molecular beacons (MBs) to detect amplified sequences containing mutations or single-nucleotide polymorphisms (SNPs). MBs have been adapted for use with the isothermal nucleic acid amplification technology loop-mediated amplification (LAMP) by targeting single-stranded loop sequences under optimized conditions to enable applications such as plant genotyping. LAMP has several benefits over PCR, such as rapid amplification, single-temperature reaction conditions enabling low-cost equipment, and robustness to certain PCR inhibitors. However, and despite the increased number of primers required, the specificity of LAMP is limited, and false positive results can be problematic. In this chapter, design considerations for molecular beacons in LAMP assays are described, as well as a method for MB-LAMP amplification and detection, with an example of gene sequences in genetically modified (GM) maize samples.

Additive manufacturing leveraged microfluidic setup for sample to answer colorimetric detection of pathogens.

Colorimetric readout for the detection of infectious diseases is gaining traction at the point of care/need owing to its ease of analysis and interpretation, and integration potential with highly specific loop-mediated amplification (LAMP) assays. However, coupling colorimetric readout with LAMP is rife with challenges including, rapidity, inter-user variability, colorimetric signal quantification, and user involvement in sequential steps of the LAMP assay, hindering its application. To address these challenges, for the first time, we propose a remotely smartphone-operated automated setup consisting of (i) an additively manufactured microfluidic cartridge, (ii) a portable reflected-light imaging setup with controlled epi-illumination (PRICE) module, and (iii) a control and data analysis module. The microfluidic cartridge facilitates sample collection, lysis, mixing of amplification reagents stored on-chip, and subsequent isothermal heating for initiation of amplification in a novel way by employing tunable elastomeric chambers and auxiliary components (heaters and linear actuators). PRICE offers a new imaging setup that captures the colorimetric change of the amplification media over a plasmonic nanostructured substrate in a controlled and noise-free environment for rapid minute-scale nucleic acid detection. The control and data analysis module employs microprocessors to automate cartridge operation in tandem with the imaging module. The different device components were characterized individually and finally, as a proof of concept, SARS-CoV-2 wild-type RNA was detected with a turnaround time of 13 minutes, showing the device's clinical feasibility. The suggested automated device can be adopted in future iterations for other detection and molecular assays that require sequential fluid handling steps.

Fully Automated CRISPR-LAMP Platform for SARS-CoV-2 Delta and Omicron Variants.

Integrated clustered regularly interspaced short palindromic repeat (CRISPR)-loop-mediated amplification (LAMP) technology is of great importance in CRISPR-based diagnostic systems, which urgently needs to be developed to improve diagnostic accuracy. A labor-free, contamination-free, and fully automated droplet manipulation platform for the CRISPR-LAMP technology has not been developed before. Herein, we propose a fully automated CRISPR-LAMP platform, which can precisely manipulate the CRISPR-LAMP droplet and perform combined reactions with high sensitivity and specificity. SARS-CoV-2 Spike T478K, D614G, P681R, and P681H mutations, typical point mutations of B.1.617.2 (Delta) and Omicron variants, are monitored with this platform with a detection limit of 102 copies/μL. Allele discrimination between the mutants and wild type is significant with the designed one/two-mismatch CRISPR RNA (crRNA) at a limit of 102 copies/μL. Chemically synthesized and modified crRNAs greatly increase the CRISPR-LAMP signal, which advance the wide application. Combined with the previously developed RdRp CRISPR-LAMP assay, clinical results showed that Spike T478K and P681H can discriminate the mutant type form the wild type with 70% (49.66-85.50%, 95% confidence interval) and 78% (57.27-90.62%, 95% confidence interval) sensitivity, respectively, and 100% specificity (51.68-100%, 95% confidence interval), and the RdRp target can detect SARS-CoV-2 strains with 85% sensitivity (65.39-95.14%, 95% confidence interval) and 100% specificity (51.68-100%, 95% confidence interval). We believe that this automatic digital microfluid (DMF) system can advance the integrated CRISPR-LAMP technology with higher stability, sensitivity, and practicability, also for other CRISPR-associated diagnostic platforms.