Isothermal Amplification Technique Research Articles

Reverse transcriptase recombinase polymerase amplification for detection of tomato spotted wilt orthotospovirus from crude plant extracts

Virus detection in early stages of infection could prove useful for identification and isolation of foci of inoculum before its spread to the rest of susceptible individuals via vectoring insects. However, the low number of viruses present at the beginning of infection renders their detection and identification difficult and requires the use of highly sensitive laboratory techniques that are often incompatible with a field application. To obviate this challenge, utilized Recombinase Polymerase Amplification, an isothermal amplification technique that makes millions of copies of a predefined region in the genome, to detect tomato spotted wilt orthotospovirus in real time and at the end point. The reaction occurs isothermically and can be used directly from crude plant extracts without nucleic acid extraction. Notably, a positive result can be seen with the naked eye as a flocculus made of newly synthesized DNA and metallic beads. The objective of the procedure is to create a portable and affordable system that can isolate and identify viruses in the field, from infected plants and suspected insect vectors, and can be used by scientists and extension managers for making informed decisions for viral management. Results can be obtained in situ without the need of sending the samples to a specialized lab.

Accurate and ultrasensitive detection for PEDV based on photoelectrochemical sensing coupling loop-mediated isothermal amplification.

Porcine epidemic diarrhea (PED) is a serious disease requiring a simple and accurate detection method. Accordingly, this study developed a novel, ultrasensitive photoelectrochemical (PEC) sensing platform using the loop-mediated isothermal amplification (LAMP) technique (LAMP-PEC). An amino (-NH2)-modified LAMP product is obtained by amplification of the PED virus gene with specially designed primers. The generated NH2-modified LAMP product is assembled on the surface of an electrode by forming imine linkages between aldehyde and amino groups based on the Schiff base reaction. A stable photocurrent is provided by a CdIn2S4 photoactive material, which possesses high photoelectric conversion efficiency. Amplified DNA assembled on the electrode surface increases steric hindrance and hinders electrons from moving from the electrode to electron acceptors, which decreases the photocurrent. This strategy can detect PEDV with a low detection limit of 0.3fgμL-1 and a wide linear range of 1×10-3-1×102pg/μL. The sensing platform has excellent specificity and sensitivity and can be used for the quantitative detection of many other pathogens with the assistance of LAMP.

Detection of chronic toxoplasmosis in the brain of mice using loop-mediated isothermal amplification (LAMP) and conventional PCR

BackgroundToxoplasma gondii is a common protozoan parasite that infects approximately one-third of the world's population. It is a disease with multiple manifestations. In immunocompetent individuals, symptoms are mild and flu-like, whereas, in immunocompromised patients, it often results in severe morbidity and mortality. Thus, studies for developing a simple, rapid diagnostic tool for early detection of Toxoplasma are emerging. Molecular diagnosis is highly accurate and helpful in congenitally infected and immunocompromised patients. The loop-mediated isothermal amplification (LAMP) technique was invented to improve nucleic acid amplification efficacy in terms of sensitivity and specificity. Aim of the studyThe study aimed to validate a LAMP protocol for detecting Toxoplasma DNA in the brain homogenates from mice experimentally infected with Toxoplasma's ME-49 (cyst-forming type II) strain in comparison to PCR. MethodsIn this study, the target DNA fragment was the Toxoplasma 529-bp, repeated 200–300 copies/genome. The sensitivity of both LAMP and conventional PCR techniques was estimated in brain homogenates in experimental mice at eight weeks post-infection and compared to the histopathology data. ResultsThe LAMP reaction showed positive results in 18 of the 26 examined samples of brain homogenates. PCR showed the characteristic 529-bp band in 15 of the 26 examined samples. ConclusionThe LAMP showed a higher sensitivity over PCR in detecting Toxoplasma infection in brain homogenates of infected mice.

Application of loop-mediated isothermal amplification to detect Salmonella spp. in egg samples

The objective of the study was to evaluate the use of loop-mediated isothermal amplification (LAMP) technique for detection of Salmonella spp. in chicken eggs. Eight Salmonella strains and 26 non-Salmonella enteric strains were used to verify the specificity of the LAMP assay for detection of Salmonella. The Sal4 primer set was used to specifically amplify the target sequence on the invA gene of Salmonella and the hydroxyl naphthol blue was used as the indicator to read the reaction results. Blank egg samples were spiked with serial 10-fold diluted Salmonella suspensions to determine the limit of detection at 50% (LOD50) of the assay. Egg samples collected from multiple locations in Ho Chi Minh City were tested for the presence of Salmonella using both LAMP method and the traditional culture method to compare the effectiveness of these two methods. Among 34 bacterial strains tested by LAMP assay, false-positive or false-negative results were not observed. The assays of spiked egg samples showed that the LOD50 of the LAMP method was less than 2 CFU/25 g sample. These results indicated a high specificity and sensitivity of the LAMP assay in detection of Salmonella in egg samples, even with those of low levels of contamination. Upon testing collected egg samples (n = 42), the LAMP assay produced the same results of Salmonella detection as the culture method. Salmonella was detected in 4 out of 42 samples. This study showed that the LAMP method is highly effective and would be suitable for use in detection of Salmonella in egg samples. The assay has several advantages such as saving time and labor compared to traditional culture methods.

Isothermal nucleic acid amplification and its uses in modern diagnostic technologies.

Nucleic acids are prominent biomarkers for diagnosing infectious pathogens using nucleic acid amplification techniques (NAATs). PCR, a gold standard technique for amplifying nucleic acids, is widely used in scientific research and diagnosis. Efficient pathogen detection is a key to adequate food safety and hygiene. However, using bulky thermal cyclers and costly laboratory setup limits its uses in developing countries, including India. The isothermal amplification methods are exploited to develop miniaturized sensors against viruses, bacteria, fungi and other pathogenic organisms and have been applied for in situ diagnosis. Isothermal amplification techniques have been found suitable for POC techniques and follow WHO's ASSURED criteria. LAMP, NASBA, SDA, RCA and RPA are some of the isothermal amplification techniques which are preferable for POC diagnostics. Furthermore, methods such as WGA, CPA, HDA, EXPAR, SMART, SPIA and DAMP were introduced for even more accuracy and robustness. Using recombinant polymerases and other nucleic acid-modifying enzymes has dramatically broadened the detection range of target pathogens under the scanner. The coupling of isothermal amplification methods with advanced technologies such as CRISPR/Cas systems, fluorescence-based chemistries, microfluidics and paper-based sensors has significantly influenced the biosensing and diagnosis field. This review comprehensively analyzed isothermal nucleic acid amplification methods, emphasizing their advantages, disadvantages and limitations.

Multiplexed Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Nucleic Acid-Based Lateral Flow Dipstick as a Rapid Diagnostic Method to Detect SARS-CoV-2.

Due to the high reproduction rate of COVID-19, it is important to identify and isolate infected patients at the early stages of infection. The limitations of current diagnostic methods are speed, cost, and accuracy. Furthermore, new viral variants have emerged with higher rates of infectivity and mortality, many with mutations at various primer binding sites, which may evade detection via conventional PCR kits. Therefore, a rapid method that is sensitive, specific, and cost-effective is needed for a point-of-care molecular test. Accordingly, we developed a rapid molecular SARS-CoV-2 detection kit with high specificity and sensitivity, RT-PCR, taking advantage of the loop-mediated isothermal amplification (LAMP) technique. Four sets of six primers were designed based on conserved regions of the SARS-CoV-2 genome: two outer, two inner and two loop primers. Using the optimized protocol, SARS-CoV-2 genes were detected as quickly as 10 min but were most sensitive at 30 min, detecting as little as 100 copies of template DNA. We then coupled the RT-LAMP with a lateral flow dipstick (LFD) for multiplex detection. The LFD could detect two genic amplifications on a single strip, making it suitable for multiplexed detection. The development of a multiplexed RT-LAMP-LFD reaction on crude VTM samples would be suitable for the point-of-care diagnosis of COVID-19 in diagnostic laboratories as well as in private homes.

Evaluation of the effect of outer primer structure, and inner primer linker sequences, in the performance of Loop-mediated isothermal amplification

Loop-mediated isothermal amplification, or LAMP, is nowadays the most popular isothermal nucleic acid amplification technique. This technique implements a minimum of four primers, named outer (F3/B3) and inner primers (FIP/BIP). The inner primers hybridize in two distinct regions, and some studies have reported that the usage of a linker, typically composed of four thymines, in the middle of these primers can improve assay performance. In addition to this, dual-priming oligonucleotides, DPO, have been reported to provide highly specific reducing non-specific amplifications. Considering the large number of primers implemented in LAMP assays, in the current study the suitability of DPO primers replacing regular outer primers; and their combination with different linker sequences in the inner primers were explored. The results demonstrated that replacing standard F3/B3 by DPO primers does not significantly affect that overall performance of the assay, and provides additional stability to temperature changes. This observations were consistent regardless the type of linker implemented in the inner primers, out of which in the current study a linker composed of thymines significantly outperformed the other options tested, most likely due to a combination of sequence and physical structure.

Reverse Transcriptase Enzyme-assisted recombinant isothermal amplification with CRISPR/Cpf1 for RNA virus assay

Foot-and-mouth disease virus (FMDV) causes Foot-and-mouth disease, which was listed as a reported disease by Office international des épizooties. It has caused great economic losses to the meat industry. Here, we developed a sensitive, rapid, and visible method based Reverse Transcriptase Enzyme-assisted recombinant isothermal amplification technique (RT-ERA) and CRISPR/Cpf1 for detecting FMDV. RT-ERA achieved simultaneous reverse transcription and amplification in a short time at 37 ℃. Moreover, CRISPR/Cpf1 specifically recognized the products and amplified the signals through the trans-cleavage activity, which converted the DNA signals into fluorescence signals and lateral flow assay strip. Within 30 min, the method detected FMDV as low as 10 copies·μL−1, making it 103 times more sensitive than gel electrophoresis and 10 times more sensitive than reverse transcription quantitative real-time PCR. Moreover, the method had good performance in actual samples. Therefore, this method realized visual detection without the large instruments and showed significant potential for FMDV assay.

Rapid Detection of Pyrethroid Resistance-Associated Mutation L1014F in the Field Based on Loop-Mediated Isothermal Amplification (LAMP) Technique

Rapid Detection of Pyrethroid Resistance-Associated Mutation L1014F in the Field Based on Loop-Mediated Isothermal Amplification (LAMP) Technique

Development and application of a dual ERA method for the detection of Feline Calicivirus and Feline Herpesvirus Type I

Feline calicivirus (FCV) and feline herpesvirus type I (FHV-1) are the most common viral pathogens responsible for cat respiratory diseases, and coinfection with these two pathogens is often found. In veterinary clinics, the main diagnostic methods for FCV and FHV-1 are test strips and polymerase chain reaction (PCR). However, the sensitivity of test strips are not sufficient, and PCR is time-consuming. Therefore, developing a rapid and high-performance clinical diagnostic test is imperative for the prevention and treatment of these diseases. Enzymatic recombinase amplification (ERA) is an automated isothermal nucleic acid amplification technique that maintains a constant temperature, and is both rapid and highly accurate. In this study, a dual ERA method was developed using the Exo probe for a differential detection of FCV and FHV-1. This dual ERA method demonstrated high performance with the detection limit of 101 copies for both viruses, and no cross-reactions with feline parvovirus virus and F81 cells. To test the utility of the method for clinical applications, 50 nasopharyngeal swabs from cats with respiratory symptoms were collected and tested. The positive rates of FCV and FHV-1 were 40% (20/50, 95% confidence interval [CI], 26.4 to 54.8%) and 14% (7/50, 95% CI, 5.8 to 26.7%), respectively. The rate of coinfection with FCV and FHV-1 was 10% (5/50, 95% CI, 3.3 to 21.8%). These results were in agreement with those found using quantitative real-time PCR. Therefore, this dual ERA method is a novel and efficient clinical diagnostic tool for FCV and FHV-1 detection.

453 Rapid SARS-CoV-2 testing with duplexed recombinase polymerase amplification and a bacteriophage internal control

OBJECTIVES/GOALS: Current COVID-19 rapid molecular tests require cartridge-reader detection, expensive circuitry, and complex microfluidics making the most accurate tests unavailable to the masses. Here we present a rapid molecular diagnostic leveraging isothermal amplification and paper-based microfluidics for a low-cost ultra-sensitive COVID-19 assay. METHODS/STUDY POPULATION: We designed a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of SARS-CoV-2 and bacteriophage MS2 RNA. RT-RPA is a sequence specific, ultrasensitive, rapid isothermal DNA amplification technique that is well suited to home based testing due to its rapid assay time, robustness, ease of use, and readout options. RT-RPA reagents are added to a tube and incubated at 39°C in a fluorometer. Realtime fluorometer data gives results in under 15 minutes. This assay also provides visual detection via lateral flow readout with results in 23 minutes. RESULTS/ANTICIPATED RESULTS: We have developed a rapid multiplexed nucleic acid amplification assay with an internal process control for SARS-CoV-2 using single-pot RT-RPA. We screened 21 primer combinations to select primers that demonstrated excellent performance and target specificity against common respiratory viruses. We demonstrate the ability to multiplex SARS-CoV-2 and MS2 detection, utilizing MS2 as an internal process control for lysis, reverse transcription, amplification, and readout. We show duplexed detection using both fluorescence readout and visual readout using lateral flow strips. Duplexed fluorescence detection shows a limit of detection of 25 copies per reaction. Duplexed lateral flow readout shows a limit of detection of 50 copies per reaction DISCUSSION/SIGNIFICANCE: We developed a duplexed RT-RPA assay for SARS-CoV-2 with fluorescence or lateral flow readout. Our assay does not re-quire expensive reader, circuity, or fluid handling. The low material cost, temperature, and robustness make it ideal for a more accurate home-based COVID-19 diagnostic.

Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation.

Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/μL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing.

Cloning the gene fragment encoding botulinum neurotoxin serotype A and establishment of a detection method for Clostridium botulinum using LAMP technique

The neurotoxins produced by Clostridium botulinum are lethal in minute amounts. Botulinum neurotoxin serotype A accounts for approximately 60% of botulism cases worldwide. Bacterial and toxin detection methods including the gold standard mouse bioassay and routine culture both require tedious procedures and need days to obtain results. Importantly, the development of a rapid and accurate technique to identify the toxin-encoding gene is crucial for timely diagnosis and treatment. This study aimed to clone a gene fragment encoding botulinum neurotoxin serotype A to serve as a positive control and establish a method based on the loop-mediated isothermal amplification (LAMP) technique to rapidly detect the encoding bont/A gene. The LAMP reaction was performed at the optimized temperature of 65˚C for 30 minutes with the in-house recombinant plasmid carrying a 250-bp fragment as the positive control. The developed method showed positive results for all strains provided by the National Institute of Hygiene and Epidemiology, consistent with the conventional polymerase chain reaction (PCR) results using the Vietnam standard with a total time roughly four times faster than the PCR amplification.

Development and application of crude sap-based recombinase polymerase amplification assay for the detection and occurrence of grapevine geminivirus A in Indian grapevine cultivars.

Geminiviruses are known to infect several fields and horticultural crops around the globe. Grapevine geminivirus A (GGVA) was reported in the United States in 2017, and since then, it has been reported in several countries. The complete genome recovered through high-throughput sequencing (HTS)-based virome analysis in Indian grapevine cultivars had all of the six open reading frames (ORFs) and a conserved nonanucleotide sequence 5'-TAATATTAC-3' similar to all other geminiviruses. Recombinase polymerase amplification (RPA), an isothermal amplification technique, was developed for the detection of GGVA in grapevine samples employing crude sap lysed in 0.5 M NaOH solution and compared with purified DNA/cDNA as a template. One of the key advantages of this assay is that it does not require any purification or isolation of the viral DNA and can be performed in a wide range of temperatures (18°C-46°C) and periods (10-40 min), which makes it a rapid and cost-effective method for the detection of GGVA in grapevine. The developed assay has a sensitivity up to 0.1 fg μl-1 using crude plant sap as a template and detected GGVA in several grapevine cultivars of a major grapevine-growing area. Because of its simplicity and rapidity, it can be replicated for other DNA viruses infecting grapevine and will be a very useful technique for certification and surveillance in different grapevine-growing regions of the country.

Rapid, sensitive, and specific detection of SARS-CoV-2 in nasopharyngeal swab samples of suspected patients using a novel one-step loop-mediated isothermal amplification (one-step LAMP) technique

BackgroundIn the absence of effective antiviral drugs or vaccines, early and accurate detection of SARS-CoV-2 infection is essential to the COVID-19 pandemic. This study developed and evaluated a novel rapid One-Step LAMP assay to directly detect the SARS-CoV-2 RNA from nasopharyngeal (NP) swab samples of patients with suspected SARS-CoV-2 infection living in deprived areas in comparison to One-Step Real-time PCR.MethodsTwo hundred fifty-four NP swab samples from patients suspected of COVID-19 infection living in deprived western areas of Iran were tested by TaqMan One-Step RT-qPCR and fast One-Step LAMP assays. Tenfold serial dilutions of SARS-CoV-2 RNA standard strain where the viral copy number in each dilution was previously determined using the qPCR and various templates were used to investigate the analytical sensitivity and specificity of the One-Step LAMP assay in triplicate. Also, the efficacy and reliability of the method compared to TaqMan One-Step RT-qPCR were evaluated using SARS-CoV-2 positive and negative clinical samples.ResultsThe results of the One-Step RT-qPCR and One-Step LAMP tests were positive in 131 (51.6%) and 127 (50%) participants, respectively. Based on Cohen’s kappa coefficient (κ), the agreement between the two tests was 97%, which was statistically significant (P < 0.001). The detection limit for the One-Step LAMP assay was 1 × 101 copies of standard SARS-CoV-2 RNA per reaction in less than an hour in triplicates. Negative results in all samples with non-SARS-CoV-2 templates represent 100% specificity.ConclusionsThe results showed that the One-Step LAMP assay is an efficient consistent technique for detecting SARS-CoV-2 among suspected individuals due to its simplicity, speed, low cost, sensitivity, and specificity. Therefore, it has great potential as a useful diagnostic tool for disease epidemic control, timely treatment, and public health protection, especially in poor and underdeveloped countries.

Colorimetric detection of SARS‐CoV‐2 variants with paper‐based analytical devices

Colorimetric detection of SARS‐CoV‐2 variants with paper‐based analytical devices

Rapid whole genome sequencing methods for RNA viruses.

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.

Rapid Detection of A282S Mutation in the RDL1 Gene of Rice Stem Borer via the Mutation-Specific LAMP Technique

Rice stem borer Chilo suppressalis (Walker) is one of the most serious pests on rice and is distributed worldwide. With the long-term and continuous usage of insecticides, C. suppressalis has developed high levels of resistance to various kinds of insecticides, including phenylpyrazole insecticides. As is well known, the resistance of C. suppressalis to phenylpyrazole insecticides is determined by the A282S mutation of the GABA receptor RDL subunit. In order to efficiently detect the resistance of C. suppressalis, a rapid and sensitive loop-mediated isothermal amplification (LAMP) technique was established and optimized in this study. The optimal concentration of components was Bst DNA polymerase (0.24 U/μL), dNTP (0.8 mM), Mg2+ (4 mM), betaine (0.6 M), forward inner primer and backward inner primer (1.6 μM), F3 and B3 (0.4 μM), and hydroxyl naphthol blue (150 mM), respectively, and the optimal reaction condition was 63 °C for 60 min, which could reduce the cost and time of detection. In addition, the accuracy of the optimized LAMP reaction system and parameters was verified in the field strains of C. suppressalis from different regions, including Jiangsu, Jiangxi, and Hu’nan provinces. The mutation (A2’S) was successfully detected in the field strains. As far as we know, this is the first report of the LAMP technique applied in the resistance monitoring of C. suppressalis to phenylpyrazole insecticides. According to our results, the optimized LAMP reaction system is feasible and easy to operate and to efficiently detect resistance-related mutation in a short time, as directly judged by the naked eye. Our results provide a new tool for detection of resistance of C. suppressalis, which is a very useful tool for comprehensive management of C. suppressalis.

AutoPLP: A Padlock Probe Design Pipeline for Zoonotic Pathogens.

Emergence of novel zoonotic infections among the human population has increased the burden on global healthcare systems to curb their spread. To meet the evolutionary agility of pathogens, it is essential to revamp the existing diagnostic methods for early detection and characterization of the pathogens at the molecular level. Padlock probes (PLPs), which can leverage the power of isothermal nucleic acid amplification techniques (NAAT) such as rolling circle amplification (RCA), are known for their high sensitivity and specificity in detecting a diverse pathogen panel of interest. However, due to the complexity involved in deciding the target regions for PLP design and the need for optimization of multiple experimental parameters, the applicability of RCA has been limited in point-of-care testing for pathogen detection. To address this gap, we have developed a novel and integrated PLP design pipeline named AutoPLP, which can automate the probe design process for a diverse pathogen panel of interest. The pipeline is composed of three modules which can perform sequence data curation, multiple sequence alignment, conservation analysis, filtration based on experimental parameters (Tm, GC content, and secondary structure formation), and in silico probe validation via potential cross-hybridization check with host genome. The modules can also take into account the backbone and restriction site information, appropriate combinations of which are incorporated along with the probe arms to design a complete probe sequence. The potential applications of AutoPLP are showcased through the design of PLPs for the detection of rabies virus and drug-resistant strains of Mycobacterium tuberculosis.

Rolling Circle Amplification on a Bead: Improving the Detection Time for a Magnetic Bioassay.

Detection of pathogens has become increasingly important, especially in the face of outbreaks and epidemics all over the world. Nucleic acid detection techniques provide a solid base to detect and identify pathogens. In recent years, magnetic sensors and magnetic labels have become of more interest due to their simplicity of use, low cost, and versatility. In this work, we have used the isothermal DNA amplification technique of rolling circle amplification (RCA) in combination with oligo-functionalized magnetic nanoparticles. Detection of RCA products takes place through specific binding between magnetic nanoparticles and RCA products. Upon binding, the relaxation frequency of the nanoparticle changes. This change was measured using an AC susceptometer. We showcase that the RCA time can be reduced for a quicker assay when performing the RCA on the surface of micrometer-sized beads, which consequently increases the hydrodynamic volume of the RCA products. This, in turn, increases the Brownian relaxation frequency shift of the nanoparticles upon binding. We performed optimization work to determine the ideal quantity of micrometer-sized particles, oligo-functionalized nanoparticles, and the amplification time of the RCA. We show that the detection of 0.75 fmol of preamplification synthetic target is possible with only 20 min of amplification time. Finally, we showcase the high specificity of the assay, as the functionalized nanoparticles are unable to bind to amplified DNA that does not match their labels. Overall, this paves the way for a simple bioassay that can be used without expensive laboratory equipment for detection of pathogens in outbreak settings and clinics around the world.