Loop-mediated Isothermal Amplification Method Research Articles

Development of a multiplex one-tube loop-mediated isothermal amplification method for rapidly simultaneous detection of the mecA and nuc genes in methicillin-resistant Staphylococcus aureus

This study developed a multiplex one-tube loop-mediated isothermal amplification method using fluorescent dye-labeled DNA probes (mfLAMP) for the rapid simultaneous detection of the nuc (S. aureus-specific marker) and mecA genes (PBP2a-specific marker) in methicillin-resistant Staphylococcus aureus (MRSA). In the mfLAMP method, a graphene oxide (GO) was used as an effective adsorbent to eliminate free fluorescent dye-labeled DNA probes (mecAf-probe: Cy5-ssDNA-Biotin and nucf-probe: FAM-ssDNA-Biotin) present in negative results. The samples were then measured using a developed program that enable simultaneous detection of fluorescein (FAM) and Cyanine5 (Cy5). Green fluorescence represents the detection of the nuc gene, red fluorescence indicates the detection of the mecA gene, and yellow fluorescence signifies the detection of MRSA. The mfLAMP was specific to MRSA and could detect 10°CFU/mL of MRSA in standard buffer and artificially inoculated irrigation water. The recovery rate from contaminated irrigation water ranged from 100.41 to 109.85 %. This study is the first to report the mfLAMP using GO, which could be point-of-care testing for the rapid and simultaneous detection of MRSA's nuc and mecA genes. The results demonstrate that the mfLAMP will be a novel tool for simultaneously detecting more than two target genes or bacteria within 1 h without an enrichment step.

Isolation of Vibrio cholerae and Vibrio vulnificus from Estuarine Waters, and Genotyping of V. vulnificus Isolates Using Loop-Mediated Isothermal Amplification

Bacteria in the genus Vibrio are ubiquitous in estuarine and coastal waters. Some species (including Vibrio cholerae and Vibrio vulnificus) are known human pathogens causing ailments like cholera, diarrhea, or septicemia. Notably, V. vulnificus can also cause a severe systemic infection (known as vibriosis) in eels raised in aquaculture facilities. Water samples were periodically collected from the estuary of the Asahi River, located in the southern part of Okayama City, Japan. These samples were directly plated onto CHROMagar Vibrio plates, and colonies displaying turquoise-blue coloration were selected. Thereafter, polymerase chain reaction was used to identify V. cholerae and V. vulnificus. A total of 30 V. cholerae strains and 194 V. vulnificus strains were isolated during the warm season when the water temperature (WT) was higher than 20 °C. Concurrently, an increase in coliforms was observed during this period. Notably, V. vulnificus has two genotypes, designated as genotype 1 and genotype 2. Genotype 1 is pathogenic to humans, while genotype 2 is pathogenic to both humans and eels. The loop-mediated isothermal amplification method was developed to rapidly determine genotypes at a low cost. Of the 194 strains isolated, 80 (41.2%) were identified as genotype 1 strains. Among the 41 strains isolated when the WTs were higher than 28 °C, 25 strains (61.0%) belonged to genotype 1. In contrast, of the 32 strains isolated when the WTs were lower than 24 °C, 27 strains (84.4%) belonged to genotype 2. These results suggest that the distribution of the two genotypes was influenced by WT.

Evaluation of the SARS-CoV-2 RNA detection reagent LAMPdirect Genelyzer KIT using nasopharyngeal swab and saliva samples

The LAMPdirect Genelyzer KIT allows for the detection of SARS-CoV-2 RNA in saliva samples with a loop-mediated isothermal amplification (LAMP) method and generates results within 20 min. It has been approved by the Pharmaceuticals and Medical Devices Agency in Japan. In this study, the performance of the LAMPdirect Genelyzer KIT was compared with that of the RT-qPCR reference method using 50 nasopharyngeal swabs and 100 saliva samples. In addition, we evaluated the applicability of an alternative reverse transcriptase and the effects of an inactivation buffer. The total agreement rates were 80.0 % and 82.0 % for nasopharyngeal and saliva samples, respectively. When considering samples at the detection limit (50 copies/reaction) that increases the chance of transmission between humans, the total agreement rates were 100% and 94.1% for nasopharyngeal and saliva samples, respectively. The LAMP method is simple, fast, and inexpensive, making it useful for small medical institutions or rural areas.

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.

Development of nicotiana-specific molecular markers and their application in a loop-mediated isothermal amplification assay

An effective identification method for detecting illegal goods involving raw tobacco material is crucial for tobacco monopolies to conduct surveillance. We developed Nicotiana-specific molecular markers to determine whether seized goods contain raw tobacco material. The sequence data for genes related to the nicotine metabolism pathway and genomic data from the public Solanaceae database were used to establish Nicotiana-specific molecular markers. These markers were determined by experimentally verifying 17 types of nontobacco plant material and 91 types of tobacco material belonging to 11 sections of 3 subgenera. Two reliable Nicotiana-specific markers, Ntsp027 and Ntsp151, were selected from among the 209 newly developed markers. The results indicated that the primers corresponding to these two markers can amplify the target fragments in the 91 types of Nicotiana material without amplification of any PCR products in the 17 types of non-Nicotiana material. Furthermore, utilizing the marker Ntsp151, we verified the efficacy of the loop-mediated isothermal amplification (LAMP) assay in authenticating tobacco material. The identification of 21 tea-cigarette products via the combination of GC‒MS, a Nicotiana-specific molecular marker and LAMP methods underscores the utility of Nicotiana-specific DNA markers in determining whether illegal goods contain raw tobacco material. Our results indicate an impressive accuracy rate of 100%, which is consistent with the reliability assessment, underscoring the accuracy of these markers in effectively identifying tobacco material. Our findings can significantly augment the capacity for surveillance and anticounterfeiting efforts by aiding the fight against illicit trade and ensuring the integrity of all tobacco-related products in the market.

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Evaluation of a rapid Loop Mediated Isothermal Amplification (LAMP) test for the laboratory diagnosis of sexually transmitted infections.

Sexually transmitted infections (STIs) have seen a considerable increase in the last years and given the health burden they may represent from both a personal and community perspective, they require surveillance and prevention programmes based on a timely and decentralized diagnosis. In this context, user-friendly rapid molecular tests may represent a good trade-off between diagnostic accuracy, accessibility and affordability. In this study we evaluated the diagnostic performance of a new real-time LAMP (Loop Mediated Isothermal Amplification) method for the rapid detection and differentiation of 7 major sexually transmissible pathogens by analysing real clinical samples (genital and extra-genital matrices) from individuals with suspected STIs. The assay showed good overall diagnostic performances in terms of sensitivity, specificity and concordance with a gold-standard PCR-based molecular method. This assay, not requiring specialised laboratory technicians or expensive instrumentation, but nonetheless capable of guaranteeing accurate results, is within the reach of outpatient settings, obstetrics, and gynaecology clinic, hence ensuring on-field access to early diagnosis.

Detection of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in Inactivated Fermented Milk Using Fluorescence Quantitative Loop-Mediated Isothermal Amplification

Currently, no effective method exists to detect and monitor fermentation probiotics and evaluate the quality of inactivated fermented milk. Therefore, in this study, a fluorescence quantitative loop-mediated isothermal amplification (FQ-LAMP) method was developed to detect Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. The specificity of LAMP primers for L. bulgaricus and S. thermophilus was verified using S-type amplification curves and a single peak at approximately 88.568°C and 83.704°C of the melting curves, respectively. The lowest quantification limits of FQ-LAMP for the two strains in inactivated fermented milk were 8.1 × 103 CFU/g (170 fg/μL) and 6.8 × 103 CFU/g (170 fg/μL), respectively. FQ-LAMP was used to analyse 40 inactivated fermented milk samples from six randomly selected brands. The logarithmic concentration of S. thermophilus in all products was between 7.482 and 8.936. The logarithmic concentration of L. bulgaricus ranged from 4.590 to 8.277, with no detectable L. bulgaricus in three samples. FQ-LAMP has the potential as a rapid, specific, and accurate method for detecting and monitoring L. bulgaricus and S. thermophilus in inactivated fermented milk during their shelf life.

A simple graphene oxide-based DNA purification strategy for plant pathogen detection.

The on-site molecular detection of plant pathogens is particularly important for the development of sustainable agriculture. Extracting DNA from plant tissues, microbes or coexisting environments is complex, labor-intensive and time-consuming. To facilitate this process, we propose a DNA purification strategy based on graphene oxide (GO). The excellent adsorption ability of GO was verified by visualizing changes in its microscopic surface and macroscopic mixture. To further optimize the DNA purification, we determined the optimal GO concentration and treatment time at 95 °C (2 mg mL-1 and 2 min, respectively). We confirmed that our strategy is effective on plant tissues and various microorganisms, and that the obtained DNA can be directly used for polymerase chain reaction amplification. Combining the proposed GO-based DNA purification method with the loop-mediated isothermal amplification method is superior, in terms of the required steps, time, cost and detection effect, to the cetyltrimethylammonium bromide method and a commercial kit for detecting plant pathogens. We present a feasible, rapid, simple and low-cost DNA purification method with high practical value for scientific applications in plant pathogen detection. This strategy can also provide important technical support for future research on plant-microbial microenvironments. © 2024 Society of Chemical Industry.

Design and evaluation of loop-mediated isothermal amplification for rapid detection of Enterocytozoon bieneusi

Enterocytozoon bieneusi is one of the most prevalent microsporidia species, responsible for more than 90% of human and animal microsporidiosis. Microsporidia species, particularly E. bieneusi, are frequently reported from waterborne and foodborne outbreaks. Therefore, early detection is crucial in clinics and outbreak investigations. This study aimed to design a loop-mediated isothermal amplification (LAMP) for rapid detection of E. bieneusi. Total DNA was extracted from 30 E. bieneusi –positive samples, which had been confirmed with nested PCR. LAMP primers were designed based on the identical fragment of small subunit ribosomal RNA (SSU rRNA) gene. LAMP reactions were performed at 63 °C for 60 min. The sensitivity and specificity of the assay were analyzed and the results of amplification were compared to real-time PCR. Our results showed that the LAMP assay successfully amplified 25/30 (83.3%) samples. The specificity results indicated no false positive with other microorganisms. Furthermore, the LAMP method exhibited a sensitivity (limit of detection, LoD) as low as 34 ag/μL of total DNA. Compared to the LAMP assay, real-time PCR was able to detect all 30 nested PCR-positive samples. Our findings showed that the LAMP assay was able to detect 83.3% of E. bieneusi-positive samples. Although the current assay was not able to detect all nested PCR-positive samples, the lack of need for specific instruments, rapid processes, and high specificity makes LAMP assay a suitable tool for screening.

A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane.

Nucleic acid quantification, allowing us to accurately know the copy number of target nucleic acids, is significant for diagnosis, food safety, agricultural production, and environmental protection. However, current digital quantification methods require expensive instruments or complicated microfluidic chips, making it difficult to popularize in the point-of-care detection. Paper is an inexpensive and readily available material. In this study, we propose a simple and cost-effective paper membrane-based digital loop-mediated isothermal amplification (LAMP) method for nucleic acid quantification. In the presence of DNA fluorescence dyes, the high background signals will cover up the amplicons-formed bright spots. To reduce the background fluorescence signals, a quencher-fluorophore duplex was introduced in LAMP primers to replace non-specific fluorescence dyes. After that, the amplicons-formed spots on the paper membrane can be observed; thus, the target DNA can be quantified by counting the spots. Take Vibrio parahaemolyticus DNA detection as an instance, a good linear relationship is obtained between the light spots and the copy numbers of DNA. The paper membrane-based digital LAMP detection can detect 100 copies target DNA per reaction within 30 min. Overall, the proposed nucleic acid quantification method has the advantages of a simple workflow, short sample-in and answer-out time, low cost, and high signal-to-noise, which is promising for application in resourced limited areas.

Fabrication of a Microfluidic Test Device with a 3D Printer and Its Combination with the Loop Mediated Isothermal Amplification Method to Detect Streptococcus pyogenes.

New rapid, reliable, and cost-effective alternative systems are needed for the rapid diagnosis of Streptococcus pyogenes. The aim of this study was to fabricate a microfluidic test device to detect Streptococcus pyogenes by combining the Loop-mediated isothermal amplification method via a 3D printer. Microfluidic test devices were designed in CATIA V5 Release 16 software, and data were directly transferred to a 3D printer and produced using the FDM method with biocompatible PLA filament. The S. pyogenes ATCC 19615 and different ATCC strains was used. Following identification by classical culture methods, a 0.5 McFarland suspension was prepared from the colonies, and DNA isolation was performed from this liquid by a boiling method. S. pyogenes specific speB gene was used to desing LAMP primer sets in PrimerExplorer V5 software and tested on a microfluidic device. LAMP reactions were performed on microfluidic device and on a microcentrifuge tube separately. Both results were analyzed using the culture method as the standard method to diagnostic values. Melting curve analysis of the amplicons of the LAMP reactions performed on a LightCycler 480 system to detect amplification. Among the 50 positive and 100 negative samples, only four samples were found to be false negative by LAMP reaction in a microcentrifuge tube, while eight samples were found to be false negative by LAMP reaction on a microfluidic device. Six samples were found to be false positive by the LAMP reaction in the microcentrifuge tube, while ten samples were found to be false positive by the LAMP reaction on a microfluidic chip. The sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP reactions performed in the microcentrifuge tube and on the microfluidic device were 92-84%, 94-90%, 88.46-80.77%, and 95.92-91.84%, respectively. The limit of detection (LOD) was found to be the same as 1.5 × 102 CFU/mL and the limit of quantification (LOQ) values of the LAMP reactions were performed on the microcentrifuge tube and on the microfluidic device were 2.46 × 102-7.4 × 102 CFU/mL, respectively. Cohen's kappa (κ) values of the LAMP reactions were performed on the microcentrifuge tube and on the microfluidic device were 0.620-0.705, respectively. In conclusion, our data showed that the LAMP method can be combined with microfluidic test device to detect S. pyogenes, this microfluidic device can be manufactured using 3D printers and results are close to gold standard methods. These devices can be combined with LAMP reactions to detect different pathogens where resources are limited and rapid results are required.

Different forms of loop-mediated isothermal amplification (LAMP) for the detection of human viruses

Infections caused by viruses in humans can result in serious illnesses and even death. This means that early detection and diagnosis is extremely important for the control of these diseases. Currently, the loop-mediated isothermal amplification (LAMP) is regarded as a reliable method because as well as being cost-effective, simple and innovative, it allows nucleic acids to be amplified without the need for a large laboratory management structure. Thus, LAMP assay can be viewed as a highly efficient molecular-based advanced detection technique for several pathogens, including viruses. Systems based on LAMP have the following features: isothermal amplification, reproducibility and robustness, and they can be used when there is scarce or low quality genetic material. The amplification involves making use of, a maximum set of three specifically- designed primers and a DNA polymerase with strand-shifting activity. In most cases, this technique is far superior to other molecular techniques such as, for example, the conventional polymerase chain reaction (PCR) and its variants. The main advantage of the LAMP method is its cost-effectiveness, as it can be employed by means of a thermocycler, water bath or thermoblock. In this mini-review, information is provided about some of the main variants of the LAMP technique which have been designed for the diagnosis of several human viral pathogens.

The Identification of Yak Meat Using Loop-Mediated Isothermal Amplification Method Coupled with Hydroxy Naphthol Blue for the Prevention of Food Fraud

Objective. Yak is found in the Qinghai-Tibet Plateau and represents a meat of high nutritional value and good flavor. However, the production of yak is limited, and yak meat adulteration is a growing concern in the marketplace. To protect consumer rights and prevent unfair competition, it is necessary to use an efficient assay to identify the species of yak meat rapidly and accurately being sold. Methods. Loop-mediated isothermal amplification (LAMP) combined with hydroxy naphthol blue (HNB) was used to identify potential adulterants. The specificity and sensitivity tests of yak-derived components were carried out to achieve the monitoring of yak-derived components. Results. The optimal color development was achieved with an external primer-to-internal primer ratio of 400 nmol/L : 1200 nmol/L, 1.5 mmol/L dNTP, and 0.32 U/μL Bst DNA polymerase with 5 mmol/L MgSO4 at 62°C amplification temperature. The detection sensitivity of LAMP-HNB for yak-derived DNA was up to 1 pg/μL. Conclusion. The LAMP-HNB assays provided a valuable tool for the identification of yak gene from adulterated meat. This further enabled the LAMP-HNB assay to be applicable in the identification of other meat products.

Evaluation of a novel lysis-based sample processing method to optimize Vibrio vulnificus detecting by loop-mediated isothermal amplification assay

BackgroundVibrio vulnificus exists as one of the most serious foodborne pathogens for humans, and rapid and sensitive detection methods are needed to control its infections. As an emerging method, The Loop-Mediated Isothermal Amplification (LAMP) assay has been applied to the early detection of various foodborne pathogens due to its high efficiency, but sample preprocessing still prolongs the complete detection. To optimize the detection process, our study established a novel sample preprocessing method that was more efficient compared to common methods.ResultUsing V. vulnificus as the detecting pathogen, the water-lysis-based detecting LAMP method shortened the preprocessing time to ≤ 1 min with 100% LAMP specificity; the detection limits of the LAMP assay were decreased to 1.20 × 102 CFU/mL and 1.47 × 103 CFU/g in pure culture and in oyster, respectively. Furthermore, the 100% LAMP specificity and high sensitivity of the water-lysis method were also obtained on detecting V. parahaemolyticus, V. alginolyticus, and P. mirabilis, revealing its excellent LAMP adaption with improvement in sensitivity and efficiency.ConclusionOur study provided a novel LAMP preprocessing method that was more efficient compared to common methods and possessed the practical potential for LAMP application in the future.

Prevalence of S. aureus and/or MRSA in hospitalized patients with diabetic foot and establishment of LAMP methods for rapid detection of the SCCmec gene

BackgroundPatients with diabetic feet are prone to be infected due to the impaired immune system. However, the prognostic outcome of different microbial infections remains controversial. Identification and rapid screening of the pathogenic microorganisms that pose the greatest threat to the prognosis of patients with diabetic foot infections (DFIs) is critical.MethodsClinical data were statistically analyzed, which were obtained from 522 patients with DFIs, including pathogenic bacterial culture results and treatment outcomes at the last return visit. In addition, a loop-mediated isothermal amplification (LAMP) detection method was developed to identify the prevalent subtype of methicillin-resistant Staphylococcus aureus (MRSA) in DFIs patients. This study was approved by the Ethics Committee of Nanfang Hospital (NFEC-202012-K6) and registered on ClinicalTrials.gov (NCT04916457) on June 1, 2021.ResultsWe found that the proportion of patients with infections of Staphylococcus aureus (S. aureus) and MRSA was 27.7% (145/522) and 33.7% (49/145), respectively. Additionally, the incidence of osteomyelitis was 46.9% (23/49) and amputation/disability was 40.8% (20/49) in patients with MRSA infection, which were significantly higher compared to patients with other types of bacterial infections such as methicillin-susceptible Staphylococcus aureus (MSSA). Notably, we demonstrated that the main prevalent subtype of MRSA in DFIs patients in our hospital was Staphylococcal chromosomal cassettes mec (SCCmec) type II. In addition, it only takes 1.5 h to complete the entire experimental procedure in this LAMP assay, providing high sensitivity (100%) and specificity (77.8%) in hospitalized patients with DFIs.ConclusionsWe demonstrated there is a very high rate of MRSA isolation in patients with DFIs and revealed that patients infected with MRSA are at a higher risk of developing osteomyelitis, and amputation or disability. Importantly, we have developed a method for quickly screening newly admitted patients for MRSA.

Versatility of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) from diagnosis of early pathological infection to mutation detection in organisms.

Loop-mediated isothermal amplification (LAMP) is a rapid, state-of-the-art DNA amplification technology, used primarily for the quick diagnosis and early identification of microbial infection, caused by pathogens such as virus, bacteria and malaria. A target DNA can be amplified within 30min using the LAMP reaction, taking place at a steady temperature. The LAMP method uses four or six primers to bind eight regions of a target DNA and has a very high specificity. The devices used for conducting LAMP are usually simple since the LAMP method is an isothermal process. When LAMP is coupled with Reverse Transcription (RT), it allows direct detection of RNA in a sample. This greatly enhances the efficiency of diagnosis of RNA viruses in a sample. Recently, the rampant spread of COVID-19 demanded such a rapid, simple, and cost-effective Point of Care Test (PoCT) for the accurate diagnosis of this pandemic. Loop-mediated isothermal amplification(LAMP) assays are not only used for the detection of microbial pathogens, but there are various other applications such as detection of genetic mutations in food and various organisms. In this review, various implementations of RT-LAMP techniques would be discussed.

Development of Loop-Mediated Isothermal Amplification for the Detection of Prototheca bovis Directly from Milk Samples of Dairy Cattle.

Prototheca bovis is an algal emerging pathogen in dairy farms causing refractory protothecal mastitis with increasing incidence worldwide and significant economic impact. P. bovis infects cows throughout the lactation cycle, including dry periods, and can persist in the udder and the environment for a long time. Since P. bovis does not respond to treatments with antibiotics, the suggested sanitary measure to restrict the spread is culling infected animals. A point-of-care test for early detection of the causative agent is critically needed to guide farm management and the appropriate treatment of mastitis. Loop-mediated isothermal amplification (LAMP) is a highly specific molecular method, time-saving, cost-effective and easy to perform in limited settings. This study aimed to develop a LAMP assay for P. bovis detection directly from milk samples; it was employed in conjunction with a commercial DNA extraction kit which was previously used to extract DNA from milk specimens containing microbes. The LAMP assay detected P. bovis DNA within 1h in milk samples spiked with P. bovis at a concentration of 50 cells/μL, enabling on-farm disease monitoring and decision-making based on a reliable diagnosis. The LAMP method will contribute to the accurate and rapid identification of P. bovis in asymptomatic or recurrent mastitis cases and consequently aid the implementation of targeted control measures and the reduction of losses in milk production.

Rapid Visual Detection of Methicillin-Resistant Staphylococcus aureus in Human Clinical Samples via Closed LAMP Assay Targeting mecA and spa Genes.

The emergence of antimicrobial resistance (AMR), particularly methicillin-resistant Staphylococcus aureus (MRSA), poses a significant global health threat as these bacteria increasingly become resistant to the most available therapeutic options. Thus, developing an efficient approach to rapidly screen MRSA directly from clinical specimens has become vital. In this study, we establish a closed-tube loop-mediated isothermal amplification (LAMP) method incorporating hydroxy-naphthol blue (HNB) colorimetric dye assay to directly detect MRSA from clinical samples based on the presence of mecA and spa genes. In total, 125 preidentified S. aureus isolates and 93 clinical samples containing S. aureus were sourced from the microbiology laboratory at Hamad General Hospital (HGH). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were computed based on conventional PCR. The assay demonstrated 100% specificity, 91.23% sensitivity, 0.90 Cohen Kappa (CK), 100% PPV, and 87.8% NPV for the clinical samples, while clinical isolates exhibited 100% specificity, 97% sensitivity, 0.926 CK, 100% PPV, and 88.89% NPV. Compared to cefoxitin disk diffusion, LAMP provided 100% specificity and sensitivity, 1.00 CK, and 100% for PPV and NPV. The study revealed that the closed-tube LAMP incorporating (HNB) dye is a rapid technique with a turnaround time of less than 1 h and high specificity and sensitivity.

Comparison of four isothermal amplification techniques: LAMP, SEA, CPA, and RPA for the identification of chicken adulteration

In recent years, with growing concern worldwide about meat and meat product adulteration, dozens of DNA-based isothermal amplification techniques have been developed and applied to detect meat adulteration. Thus, we evaluated four mainstream isothermal amplification techniques, including loop-mediated isothermal amplification (LAMP), denaturation bubble-mediated strand exchange amplification (SEA), cross-priming amplification (CPA), and recombinase polymerase amplification (RPA) focusing on the limit of detection, simplicity, amplification time and cost to confirm the most effective and suitable method for point-of-care testing (POCT) of chicken and chicken product authenticity. The LAMP, CPA, and RPA primers all targeted the chicken mitochondrial cytochrome b gene. The SEA primers were provided by the SEA kit. All methods showed good specificity to chicken. Among them, LAMP, CPA, and RPA have the lowest detection limit, with 1.2 pg μL−1. As low as 0.1% chicken in mutton can be detected in LAMP and RPA methods. Although RPA costs 10 times more than LAMP, the system and primers of LAMP are too complex. Therefore, it must be admitted that RPA is the most suitable method in multiplex detection, and LAMP is much better than the other three methods in single-plex detection. Our research provides an excellent reference value for the rapid on-site detection of meat and meat product adulteration.