Loop-mediated Isothermal Amplification System Research Articles

Rapid and quantitative detection of Aspergillus niger Van Tieghem using loop-mediated isothermal amplification assay

AbstractPeanut (Arachis hypogaea L.) crown rot and root rot are common diseases caused by Aspergillus niger Van Tieghem. Early and accurate detection of A. niger is key to disease management. In this study, the design of two to five sets of loop-mediated isothermal amplification (LAMP) primers was based on the EglA, GOD, Tub, NRPS, Tan, CbhA, and CbhB genes of A. niger. Of these, primer set GOD-91 was selected for optimization of the three-factor LAMP system: the Bst DNA polymerase concentration, the concentration ratio of the inner and outer primers, and the concentration of Mg2+. In addition, the optimized LAMP reaction system for A. niger detection was validated for specificity, sensitivity, and on-site feasibility. The specificity test showed that A. niger could be specifically detected with the proposed method without cross-amplification of other pathogenic fungi DNA. Moreover, based on the sensitivity test, the lowest detection limit of this reaction system was 5.1 × 10−7 ng/µL pAN01 plasmid DNA, after which a standard curve was generated for the quantitative detection of A. niger. The LAMP method was further applied for field sample assessment before and after A. niger infection, successfully detecting A. niger presence in the samples collected in the field. This study yielded a sensitive, specific, and reproducible LAMP system that can be used to assess on-site samples within 45 min. It is an effective approach for the rapid and quantitative detection of A. niger.

Rapid DNA extraction and microfluidic LAMP system in portable equipment for GM crops detection

Genetically modified food has come under suspicion because of the potential safety risks. Only a limited number of target genes could be detected by traditional methods. Here, customized-fast DNA extraction methods and a LAMP (Loop-mediated isothermal amplification) system were developed combining with a hand-held chip, a portable analyzer, and an Android App for the qualitative detection of genetically modified crops. DNA of seeds and leaves was obtained within 5 minutes. Primers were designed with high specificity and sensitivity (10 copies) for later LAMP amplification. Crude DNA was added to the chip pre-embedded with customizable primers by quantitative dropper and the whole detection process was less than 40 minutes without any large-scale instrument. The 0.1% quality percentage of GM samples (maize or soybean) could be detected by the detection system. The detection system with its low workload, high accuracy, and stability offers a novel and effective approach for the rapid detection of GM crops and many other food safety areas in the field.

Establishment and application of a loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for detecting Clostridium piliforme.

Clostridium piliforme (causative agent of Tyzzer disease) infects various animals, including primates, and hence a threat to animal and human health worldwide. At present, it is detected using traditional methods, such as path morphology, polymerase chain reaction and enzyme-linked immunosorbent assay. Therefore, it is necessary to develop convenient, efficient visual molecular biological methods for detecting C. piliforme. To establish a method with good specificity, high sensitivity and simple operation for the detection of C. piliforme. In this study, we designed internal and external primers based on the conserved 23S rRNA region of C. piliforme to develop a biotin-labelled diarrhoea-suffered loop-mediated isothermal amplification (LAMP) system for detecting of C. piliforme and assessed the specificity, sensitivity and repeatability of the LAMP system. The LAMP system did not exhibit cross-reactivity with 24 other common pathogenic species, indicating that it had good specificity. The minimum concentration of sensitivity was 1×10-7 ng/μL. Mouse models (Meriones unguiculatus) of Tyzzer disease were established and a LAMP-lateral flow dipstick (LAMP-LFD) was developed for detecting C. piliforme. The detection rate of C. piliforme was 5.08% in clean-grade animals and 9.96% in specific-pathogen-free-grade animals from Jiangsu, Zhejiang and Shanghai. In addition, the detection rates of C. piliforme were 10.1%, 8.6% and 20%, in animals from Hangzhou, Wenzhou and Shaoxing, respectively. The detection rate of C. piliforme was higher in experimental animals used in schools than in those used in companies and research institutes. The LAMP-LFD method established in this study can be used to detect C. piliforme in animals handled in laboratory facilities of universities, pharmaceutical enterprises and research and development institutions.

Isothermal Amplification and Hypersensitive Fluorescence Dual-Enhancement Nucleic Acid Lateral Flow Assay for Rapid Detection of Acinetobacter baumannii and Its Drug Resistance.

Acinetobacter baumannii (A. baumannii) is among the main pathogens that cause nosocomial infections. The ability to rapidly and accurately detect A. baumannii and its drug resistance is essential for blocking secondary infections and guiding treatments. In this study, we reported a nucleic acid fluorescent lateral flow assay (NFLFA) to identify A. baumannii and carbapenem-resistant A. baumannii (CRAB) in a rapid and quantitative manner by integrating loop-mediated isothermal amplification (LAMP) and silica-based multilayered quantum dot nanobead tag (Si@MQB). First, a rapid LAMP system was established and optimised to support the effective amplification of two bacterial genes in 35 min. Then, the antibody-modified Si@MQB was introduced to capture the two kinds of amplified DNA sequences and simultaneously detect them on two test lines of a LFA strip, which greatly improved the detection sensitivity and stability of the commonly used AuNP-based nucleic acid LFA. With these strategies, the established LAMP-NFLFA achieved detection limits of 199 CFU/mL and 287 CFU/mL for the RecA (house-keeping gene) and blaOXA-23 (drug resistance gene) genes, respectively, within 43 min. Furthermore, the assay exhibited good repeatability and specificity for detecting target pathogens in real complex specimens and environments; thus, the proposed assay undoubtedly provides a promising and low-cost tool for the on-site monitoring of nosocomial infections.

Development of a Loop-Mediated Isothermal Amplification (LAMP) Method to Detect the Potato Zebra Chip Pathogen 'Candidatus Liberibacter solanacearum' (Lso) and Differentiate Haplotypes A and B.

'Candidatus Liberibacter solanacearum' (Lso) is the causal agent of zebra chip of potato (Solanum tuberosum), which can significantly reduce potato yield. In this study, a loop-mediated isothermal amplification (LAMP) method for the detection of Lso haplotypes A and B was developed and evaluated. Two sets of LAMP primers named LAMP-A and LAMP-B were designed and tested for specificity and sensitivity. Both LAMP-A and LAMP-B were specific to Lso in in silico analysis using the Primer-Blast tool. The LAMP-A and LAMP-B could only produce positive signals from DNA mixtures of Lso-infected tomato but not from the genomic DNA of 37 nontarget plant pathogens. The sensitivity of LAMP-A and LAMP-B on Lso haplotypes A and B were tested on gBlocks and genomic DNA from Lso-infected tomato. On the genomic DNA for LAMP-A, the lowest amount of template DNA for a positive LAMP reaction was 2 to 20 ng on four haplotype A strains and 20 to 80 ng on four haplotype B strains; for LAMP-B, the lowest amount of template DNA for a positive LAMP reaction was 0.02 to 2 ng on four haplotype B strains and 20 ng to no amplification on four haplotype A strains. On gBlocks for LAMP-A, the lowest number of copies for a positive LAMP reaction was 60 on haplotype A and 600 on haplotype B; for LAMP-B, the lowest number of copies for a positive LAMP reaction was 60 on haplotype B and 600 on haplotype A. Therefore, considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, the LAMP-A and LAMP-B primers can be used together to test the probable Lso-infected plant or psyllid samples to rapidly, accurately, and directly differentiate haplotypes A and B. We highly recommend this LAMP system to plant pathology practitioners and diagnostic labs for routine detection of Lso and confirmation of zebra chip disease on potato or tomato.

Field-deployable multiplex detection method of SARS-CoV-2 and influenza virus using loop-mediated isothermal amplification and DNA chromatography.

A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings.

An optimized visual loop mediated isothermal amplification assay for efficient detection of minute virus of mice with hydroxynaphthol blue dye

Minute virus of mice (MVM) is one of the most prevalent infectious agents in laboratory mouse colonies. In this study, we optimized a loop-mediated isothermal amplification (LAMP) assay using hydroxynaphthol blue (HNB) for rapid and visual detection of MVM. The reaction, which entailed addition of HNB dye prior to amplification, was performed in one step in a single tube at 62 °C for 45 min. The limit of detection of the assay was 104 copies, which was 100-fold lower than that of conventional PCR. The assay specifically amplified MVM DNA and did not cross-react with other viruses. To validate the established LAMP system, we applied it 287 samples and detected 19 positives. In conclusion, LAMP with HNB is a sensitive, and simple assay for rapid detection of MVM infections in laboratory animals, thus offers a platform for quality monitoring.

Capping Gold Nanoparticles to Achieve a Protein-like Surface for Loop-Mediated Isothermal Amplification Acceleration and Ultrasensitive DNA Detection.

Loop-mediated isothermal amplification (LAMP) is a popular DNA amplification method. Gold nanoparticles (AuNPs) were reported to enhance the efficiency of LAMP, although the underlying mechanism remained elusive. Since AuNPs strongly adsorb a range of ligands, preadsorbed ligands cannot be easily displaced. In this work, we systematically investigated the effect of surface-modified AuNPs on LAMP by varying the order of mixing of AuNPs with each reagent in the LAMP system (Mg2+, template DNA, dNTPs, primers, and polymerase). Mixing the AuNPs with the primers delayed the LAMP based on SYBR green I fluorescence. While other orders of mixing had little effect, all accelerated the reaction. We then tested other common ligands including polymers (polyethylene glycol and polyvinylpyrrolidone), inorganic ions (Br-), proteins, glutathione (GSH), and DNA (A15) on AuNP-LAMP. The boosted AuNP performance on LAMP was most obvious when the AuNPs formed a protein-like surface. Finally, using GSH-capped AuNPs, a detection limit of around 100 copies/μL-1 of target DNA was achieved. This work has identified a ligand-capped AuNP strategy to boost LAMP and yielded a higher sensitivity in DNA sensing, which also deepens our understanding of AuNP-assisted LAMP.

Visual detection of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) simultaneously by duplex loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is often confounded by the non-specific amplification arising from primer dimers, off-target priming, and other artifacts. Precipitation of the DNA produced during LAMP with the use of specific fluorescently labeled probe has proved the effectiveness in specific detection. Herein, two fluorophores (ROX and FAM) were attached to the primers S-LB-6 and R-FIP for Atlantic salmon and rainbow trout, respectively, which are self-quenched in unbound state and become de-quenched after binding to the dumbbell-shaped DNA specifically. The DNA precipitation and appearance of small sediment took 10 s of centrifugation at 1000 g, by adding polyethylenimine (PEI) 600. Each target species was specifically amplified with the predicted color of PEI-DNA sediment, namely red for Atlantic salmon, green for rainbow trout, and pale yellow for mixed species. The optimized duplex LAMP system has proved its specificity and can detect as little as 1 ng DNA in visual detection.

Highly Specific Loop-Mediated Isothermal Amplification Using Graphene Oxide-Gold Nanoparticles Nanocomposite for Foot-and-Mouth Disease Virus Detection.

Loop-mediated isothermal amplification (LAMP) is a molecular diagnosis technology with the advantages of rapid results, isothermal reaction conditions, and high sensitivity. However, this diagnostic system often produces false positive results due to a high rate of non-specific reactions caused by formation of hairpin structures, self-dimers, and mismatched hybridization. The non-specific signals can be due to primers used in the methods because the utilization of multiple LAMP primers increases the possibility of self-annealing of primers or mismatches between primers and templates. In this study, we report a nanomaterial-assisted LAMP method that uses a graphene oxide–gold nanoparticles (AuNPs@GO) nanocomposite to enable the detection of foot-and-mouth disease virus (FMDV) with high sensitivity and specificity. Foot-and-mouth disease (FMD) is a highly contagious and deadly disease in cloven-hoofed animals; hence, a rapid, sensitive, and specific detection method is necessary. The proposed approach exhibited high sensitivity and successful reduction of non-specific signals compared to the traditionally established LAMP assays. Additionally, a mechanism study revealed that these results arose from the adsorption of single-stranded DNA on AuNPs@GO nanocomposite. Thus, AuNPs@GO nanocomposite is demonstrated to be a promising additive in the LAMP system to achieve highly sensitive and specific detection of diverse diseases, including FMD.

Retroreflection-based sandwich type affinity sensing of isothermal gene amplification products for foodborne pathogen detection.

Loop-mediated isothermal amplification (LAMP) is an outstanding method for molecular diagnostics, as the rapid, specific, and sensitive amplification of target genes is possible. However, it is necessary to measure fluorescence in the quantitative analysis of LAMP products, so a sophisticated optical setup is required. This study tried to develop a novel sensing method that can quantify target analytes with simple equipment, such as nonspectroscopic white light and a CMOS camera. To achieve this, a retroreflective Janus particle (RJP) as a probe and specially designed loop primers, fluorescein isothiocyanate (FITC)- and biotin-modified loop primers, were introduced into the LAMP system. By performing LAMP in the presence of designed primers, double-stranded amplicons possessing FITC and biotin labels at each end are generated in proportion to the quantity of the target pathogen. Using the anti-FITC antibody-modified sensing surface and streptavidin-conjugated RJP probes, the amplicons can be captured in sandwich-configuration and detected under nonspectroscopic conditions composed of white light and a camera. To confirm the feasibility of the sensing system, the invA gene of Salmonella was selected as the target. It was possible to quantitatively analyze the Salmonella concentration from 0 to 106 colony-forming units, sufficiently covering the required detection range. In addition, quantitative analyses of pathogens in contaminated food sources, including milk and chicken meat, were successfully conducted with a limit of detection of 10 CFU.

Design of Rapid Detection System for Five Major Carbapenemase Families (bla KPC, bla NDM, bla VIM, bla IMP and bla OXA-48-Like) by Colorimetric Loop-Mediated Isothermal Amplification.

PurposeCarbapenemase-producing Enterobacteriaceae (CPE) infection constitutes a public health threat. Timely and efficient diagnosis is of paramount importance for prompt and effective therapy. In order to quickly and comprehensively detect the five major families of carbapenemases (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48-like), colorimetric loop-mediated isothermal amplification (LAMP) was employed.Materials and MethodsFive sets of LAMP primers were designed, each of which can, respectively, amplify all the carbapenemase subtypes described in this work. Twenty whole genome sequencing-verified-“standard strains”, including 1 blaNDM-1, 1 blaNDM-5, 1 blaNDM-6, 1 blaNDM-7, 2 blaIMP-4, 1 blaIMP-8, 2 blaKPC-2, 1 blaKPC-3, 1 blaKPC-4, 1 blaKPC-5, 1 blaKPC-6, 1 blaKPC-7, 1 blaOXA-48 and 1 blaOXA-181 carrier, and 1 blaVIM and blaOXA-244, 1 blaKPC-2 and blaIMP-4, 1 blaKPC-2 and blaVIM-1 and 1 blaKPC-2 and blaNDM-1-co-carriers, were used to establish a 25-microliter visual LAMP reaction system (kept at 65°C for 30 minutes in water bath). Color change from bright pink to yellow indicated positive amplification. In addition, 126 pre-verified clinical carbapenem-resistant Enterobacteriaceae (CRE) isolates, including 65 CPE (23 blaNDM, 2 blaOXA-48-like, 1 blaKPC and blaVIM, 2 blaIMP, and 37 blaKPC carriers) and 61 non-CPE, were also detected.ResultsWith the lowest detection limit of 10 colony forming units (CFU) per reaction for LAMP and 103 CFU per reaction for PCR, the LAMP system demonstrated dramatically higher sensitivity while retaining the same specificity. Furthermore, we demonstrated concordant results between the two methods for the 126 clinical isolates.ConclusionTherefore, LAMP could be used for rapid identification of the five major carbapenemase gene families in routine clinical laboratories.

Comparative study of the loop-mediated isothermal amplification method and the QIAGEN therascreen PCR kit for the detection of EGFR mutations in non-small cell lung cancer.

BackgroundEpidermal growth factor receptor (EGFR) mutations are important biomarkers in the treatment of patients with advanced or metastatic diseases. The therascreen EGFR Rotor-Gene Q (RGQ) PCR Kit® (Qiagen, Inc.) is an approved diagnostic test for EGFR mutations in non-small cell lung cancer (NSCLC). This study aims to investigate the diagnostic capability of a loop-mediated isothermal amplification (LAMP) assay as an accurate, efficient, and cost-effective alternative to the therascreen assay.MethodsEGFR mutations were investigated by LAMP and therascreen assays using tissue samples that were surgically resected or biopsied from 117 consecutive patients with NSCLC tumors. The EGFR status from the LAMP assay was compared with that of the therascreen assay. Next-generation sequencing (NGS) was performed to confirm EGFR status of tumors that did not match in both assays. To establish an optimal LAMP AUC value, receiver operating characteristics (ROC) curve analysis was performed within tumors with exon 19 deletion or L858R point mutation.ResultsOf the 117 tumors assayed, 45 tumors with EGFR mutations and 68 tumors with EGFR wild type were matched in both assays, four tumors having mismatched EGFR statuses. NGS further confirmed that two of the four discordant tumors had the same EGFR status that was determined by the LAMP assay. The AUC values were 0.973 (95% CI: 0.929–1.00) in exon 19 deletion, and 0.952 (95% CI: 0.885–1.00) in L858R point mutation. In exon 19 deletion, sensitivity, specificity, and accuracy were 89.3%, 98.9%, and 96.6%, respectively, and 94.7%, 95.9%, and 95.7%, respectively, in L858R using AUC value of 0.222.ConclusionsThe LAMP assay compared favorably with the therascreen assay and has potential as an effective, simple, rapid, and low-cost diagnostic alternative. Based on these results, a liquid biopsy LAMP system should be developed for point-of-care testing of oncogenes in the near future.

New Insights from Chemical Biology: Molecular Basis of Transmission, Diagnosis, and Therapy of SARS-CoV-2

Coronavirus disease 2019 (COVID-19) is caused by a novel strain of coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has caused a global pandemic rapidly s...

Establishment of a porcine parvovirus (PPV) LAMP visual rapid detection method

Porcine parvovirus (PPV) is one of the major causes of reproductive pig disease. Due to its serious nature, wide spread and consequent great damage to the swine industry, an effective, rapid and convenient method for its detection is needed. A loop-mediated isothermal amplification (LAMP) assay was established to detect PPV infection. Two pairs of primers were specifically designed to recognize the six different sequences of open reading frame1 (ORF1) gene. The optimized LAMP program was as follows: 50 min at 59 °C followed by 3 min at 80 °C.The amplified products were analyzed both by visual inspection after staining with SYBR Green I dye and by conventional agarose gel electrophoresis. Both methods showed the same sensitivity. The limit of detection (LOD) for PPV by LAMP was 10 copies, which is 100-fold lower than conventional PCR. Our LAMP assay did not cross-react with other viruses. We used the established LAMP system to test 1100 field samples and detected 660 positives. The LAMP detection method for PPV represents a visual, sensitive and rapid assay which can detect the virus in the field, offering an attractive alternative for the PPV detection methods currently in use.

Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants.

Simple SummaryContagious agalactia (CA) is an infectious disease of small ruminants endemic in the Mediterranean countries, causing significant socioeconomic impacts predominantly on small-scale farmers who still subsist on marginal lands. Mycoplasma agalactiae is historically considered the principal etiological agent of CA, especially in sheep. Clinical signs are characterised by mastitis, arthritis, keratoconjunctivitis and occasionally, abortion. Rapid, accurate and cost-effective field tests are urgently needed for effective control of M. agalactiae mastitis. Our study illustrated the validation of a Loop-Mediated Isothermal Amplification (LAMP) test for the detection of M. agalactiae in dairy sheep in order to confirm its application as a diagnostic tool in the field level.Contagious agalactia (CA), an infectious disease of small ruminants, caused by Mycoplasma agalactiae, is responsible for severe losses to dairy sheep production with substantial socioeconomic impacts on small-scale farmers. The diagnosis of CA is still problematic, time-consuming and requires well-equipped labs for confirmation of outbreaks. Therefore, rapid, accurate and cost-effective diagnostic tests are urgently needed. This work aims to validate a novel Loop-Mediated Isothermal Amplification (LAMP) test, based on the p40 target gene, for the detection of M. agalactiae in dairy sheep in order to confirm its potential practical use as a rapid and cheap field test. The LAMP system proposed in this study consists of a portable device composed of real-time fluorometer with the automatic interpretation of results displayed in a tablet. A total of 110 milk samples (90 positives and 20 negatives) were analysed to optimise the analysis procedure and to investigate the efficacy and robustness of the LAMP method. All samples were analysed using LAMP and conventional real-time PCR to compare the diagnostic sensitivity of the methods. The sensitivity of the LAMP was 10-fold higher than that of real-time PCR, with a detection limit up to 103 CFU/ml. The LAMP assay was able to detect M. agalactiae in 81 of 90 (90%, 95%CI 0.84–0.96) positive milk samples compared to 69 (77%, 95%CI 0.59–0.95) positive samples detected by real-time PCR; no positive signal occurred for any of the negative milk samples in either test. Therefore, the LAMP assay was found to be more sensitive than real-time PCR, low-cost, easy to perform, fast and not affected by contamination, indicating its potential as an effective diagnostic tool in the field level for the diagnosis of CA.

Validation of a Commercial Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Anisakis spp. DNA in Processed Fish Products.

Parasites belonging to the Anisakis genera are organisms of interest for human health because they are responsible for the Anisakiasis zoonosis, caused by the ingestion of raw or undercooked fish. Furthermore, several authors have reported this parasite to be a relevant inducer of acute or chronic allergic diseases. In this work, a rapid commercial system based on Loop-Mediated Isothermal Amplification (LAMP) was optimised and validated for the sensitive and rapid detection of Anisakis spp. DNA in processed fish products. The specificity and sensitivity of the LAMP assay for processed fish samples experimentally infected with Anisakis spp. larvae and DNA were determined. The LAMP system proposed in this study was able to give positive amplification for all the processed fish samples artificially contaminated with Anisakis spp., giving sensitivity values equal to 100%. Specificity tests provided no amplification for the Contracaecum, Pseudoterranova, or Hysterothylacium genera and uninfected samples. The limit of detection (LOD) of the LAMP assay proposed was 102 times lower than the real-time PCR method compared. To the best of our knowledge, this is the first report regarding the application of the LAMP assay for the detection of Anisakis spp. in processed fish products. The results obtained indicate that the LAMP assay validated in this work could be a reliable, easy-to-use, and convenient tool for the rapid detection of Anisakis DNA in fish product inspection.

Development of a Loop-mediated Isothermal Amplification Assay for Rapid Detection of Streptococcus pneumoniae Isolates in Clinical Sputum Samples

Streptococcus pneumoniae is one of the leading causes of invasive infections. Traditional culture and identification methods require a long time period and are liable to be complicated by normal flora. In this study, a new method was developed based on loop-mediated isothermal amplification. A series of primers for autolysin (LytA) from S. pneumoniae were designed with a bioinformatics technique, and a loop-mediated isothermal amplification system for the detection of S. pneumoniae was set up. Comparative analysis of specificity and sensitivity from the culture and polymerase chain reaction methods in 50 samples collected from patients with respiratory infections was performed. The data demonstrated that the selected primers and reaction system in the loop-mediated isothermal amplification were respondent to Streptococcus pneumoniae and did not cross-react with other bacteria. The detection of sensitivity to the loop-mediated isothermal amplification method was 102 CFU/ml. From all of the clinical specimens tested, 9 cases (18.0 %) were confirmed to be positive for Streptococcus pneumoniae using the loop-mediated isothermal amplification method, whereas only 4 cases (8.0 %) were diagnosed using the culture method. Loop-mediated isothermal amplification technology is a specific, simple and accurate method for the efficient differentiation of S. pneumoniae in clinical specimens.

Fast and Parallel Detection of Four Ebola Virus Species on a Microfluidic-Chip-Based Portable Reverse Transcription Loop-Mediated Isothermal Amplification System.

Considering the lack of official vaccines and medicines for Ebola virus infection, reliable diagnostic methods are necessary for the control of the outbreak and the spread of the disease. We developed a microfluidic-chip-based portable system for fast and parallel detection of four Ebola virus species. The system is based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and consists of four specific LAMP primers, a disc microfluidic chip, and a portable real-time fluorescence detector. It could specifically and parallelly distinguish four species of the Ebola virus after only one sampling, including the Zaire Ebola virus, the Sudan Ebola virus, the Bundibugyo Ebola virus, and the Tai Forest Ebola virus, without cross-contamination. The limit of detection was as small as 10 copies per reaction, while the total consumption of sample and reagent was 0.94 μL per reaction. The final results could be obtained in 50 min after one addition of sample and reagent mixture. This approach provides simplicity, high sensitivity, and multi-target parallel detection at a low cost, which could enable convenient and effective on-site detections of the Ebola virus in the outdoors, remote areas, and modern hospitals.

Application of loop-mediated isothermal amplification (LAMP) for rapid detection of Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus) and haddock (Melanogrammus aeglefinus)

Codfish is a commercially important species of sea fish and plays an important role in the world fishery. In our study, two loop-mediated isothermal amplification (LAMP) assays (real-time fluorescence LAMP and visual LAMP) were established for the identification of three cod species in Gadidae (Gadus morhua, Gadus macrocephalus and Melanogrammus aeglefinus). 12S rDNA gene was used to design primers to distinguish the Gadidae and non-Gadidae species, and the mitochondrial Cytb gene was selected for discrimination of three cod species. After optimization, the 12S rDNA system and species-specific systems performed well, and target cod DNA could be detected in single or mixed samples. In the species-specific systems, the absolute limit of detection (LODa) of three cod species were 285, 37 and 197 pg/μL, and the relative limit of detection (LODr) reached to 1%, 0.1% and 1%, respectively. In the 12S rDNA system, the LODa of three cod species were 28.5, 37 and 19.7 pg/μL, respectively, and the LODr reached to 0.1%. Through the detection of 13 commercial cod products, the LAMP systems can detect cod contents in raw materials and deep-processed products as well. It indicated that the methods developed in this study have strong practicability and can meet the needs of routine testing.