Loop-mediated Isothermal Amplification Primers Research Articles

Detection of Esca‐associated fungi in grapevine trunks using loop‐mediated isothermal amplification (LAMP) assays

AbstractEsca is a grapevine trunk disease (GTD) that is caused by filamentous fungi. It is responsible for considerable economic losses in viniculture on a global scale. Despite many unknown factors contributing to the development of symptoms in affected plants, Phaeoacremonium minimum (PMI), Phaeomoniella chlamydospora (PCH) and Fomitiporia mediterranea (FMED) are generally considered as the main causative fungal species. Early detection and specific identification of these pathogens therefore play an important role in disease control and evaluation of suitable countermeasures. In this study, loop‐mediated isothermal amplification (LAMP) assays were developed for each of the three pathogens. A genome‐based approach was applied for detection and selection of unique target DNA sequences. The designed primer sets showed overall good specificities, with some observed cross‐reactions towards closely related Phaeoacremonium species for the PMI primer set. The developed assays had detection limits of 100 pg (FMED, PMI) and 1 pg (PCH) per reaction (corresponding to 1460 [FMED]; 1950 [PMI]; 342 [PCH] genome copies per reaction). The application of the assays to field samples was demonstrated by testing individual infected grapevine trunks from two European viticultural regions using crude DNA obtained in a rapid sample preparation step. LAMP assay results matched those of PCR following a conventional DNA extraction protocol. The study showed that LAMP‐based rapid molecular detection of major Esca agents can serve as a useful tool for further research and surveillance of a highly devastating grapevine disease. The application of computer‐based whole genome comparison between target and non‐target species for the identification of unique target sequences as the basis for LAMP (or PCR) primer design was demonstrated to be a useful approach in species for which scarce sequence information is available. Moreover, the developed method for rapid DNA preparation from grapevine trunks may potentially be adapted to the DNA‐based detection also of other fungal species that cause grapevine trunk diseases.

Rapid on-site detection of Leuconostoc citreum in commercially processed products using loop-mediated isothermal amplification(LAMP) technique

Leuconostoc citreum is widely utilized across numerous industries due to its beneficial properties. Thus, a detection method for this target species is essential to track the presence of L. citreum in commercial products. We developed a rapid, on-site L. citreum detection method using the loop-mediated isothermal amplification (LAMP) technique in this study. The designed LAMP primer set exhibited high specificity, only targeting the intended species among the 76 tested. Moreover, this assay demonstrated a sensitivity of 2 pg of target DNA. To evaluate the LAMP assay's applicability, we selected 20 varied types of processed products. The results confirmed these products' successful and precise detection of L. citreum within 35 min. Additionally, we used samples spiked with L. citreum to determine the detection limit, revealing L. citreum detection up to 102 or 103 CFU/mL. Thus, the LAMP assay developed herein is a specific, sensitive, precise, and rapid on-site detection method for L. citreum in various processed products.

Sensitive and rapid detection of tet(X2) ~ tet(X5) by loop-mediated isothermal amplification based on visual OTG dye

The emergence of tigecycline-resistant tet(X2/X3/X4/X5) genes poses a new threat to the efficacy of anti-infective therapy and the safety of our food and environment. To control the transfer of such genes, a sensitive and rapid molecular method is warranted to detect tet(X2/X3/X4/X5) genes in clinical isolates. Herein, we established a loop-mediated isothermal amplification (LAMP) assay to rapidly detect tet(X2/X3/X4/X5) genes, and the results were assessed by chromogenic visualization. The specificity and sensitivity of the primers during the LAMP assay for the simultaneous detection of tet(X2/X3/X4/X5) genes were determined in this study. All 48 clinical strains without tet(X2/X3/X4/X5) genes yielded negative results during the LAMP assay, substantiating the high specificity of the LAMP primers. The detection thresholds of this assay were 1.5 × 102 CFU/ml and 0.2 fg/uL corresponding to a 10 to 100-fold and 100-fold increase in sensitivity compared to polymerase chain reaction (PCR) assays. Out of 52 bacterial strains tested, using PCR as a reference, our research revealed that the LAMP assay demonstrated a sensitivity and specificity of 100%. To sum up, our novel approach has huge prospects for application in the simultaneous detection of tet(X2/X3/X4/X5) genes and can be applied to detect other drug-resistance genes.

Real-Time and Rapid Detection of Phytopythium vexans Using Loop-Mediated Isothermal Amplification Assay.

Phytopythium vexans (de Bary) Abad, de Cock, Bala, Robideau, A. M. Lodhi & Levesque is an important waterborne and soil-inhabiting oomycete pathogen causing root and crown rot of various plants including certain woody ornamentals, fruit, and forest trees. Early and accurate detection of Phytopythium in the nursery production system is critical, as this pathogen is quickly transported to neighboring healthy plants through the irrigation system. Conventional methods for the detection of this pathogen are tedious, frequently inconclusive, and costly. Hence, a specific, sensitive, and rapid molecular diagnostic method is required to overcome the limitations of traditional identification. In the current study, loop-mediated isothermal amplification (LAMP) for DNA amplification was developed for the identification of P. vexans. It was evaluated using real-time and colorimetric assays. Several sets of LAMP primers were designed and screened, but PVLSU2 was found to be specific to P. vexans as it did not amplify other closely related oomycetes, fungi, and bacteria. Moreover, the developed assays were sensitive enough to amplify DNA up to 102 fg per reaction. The real-time LAMP assay was more sensitive than traditional PCR and culture-based methods to detect infected plant samples. In addition, both LAMP assays detected as few as 100 zoospores suspended in 100 ml water. These LAMP assays are anticipated to save time in P. vexans detection by disease diagnostic laboratories and research institutions and enable early preparedness in the event of disease outbreaks.

Simultaneous and rapid colorimetric detection of distinct miRNAs using Split-LAMP.

Introduction: Aberrant microRNA (miRNA) expressions are often discovered in many life threatening diseases such as cancer. In particular, recent studies show combinations of miRNA levels have greater diagnostic accuracy as opposed to single miRNA levels. For point-of-care applications, rapid and sensitive isothermal amplification with loop-mediated isothermal amplification (LAMP) has gained significant interest. Method: We developed a cost-effective point-of-care testing (POCT) device for multiple miRNAs that can integrate miRNA signals into a single output. Results and Discussion: We demonstrate that the loop primers for LAMP can be broken and be used for miRNA detection. This split-LAMP approach provides a logic AND-gate output for two distinct miRNA inputs. We then show that this is potentially useable in point-of-care testing using pH-sensitive dye to give a rapid, colorimetric endpoint readout within 30 min. This novel logic gate approach can potentially be extended to multiple miRNAs such that there can be a powerful diagnostic concept for multiple short RNAs in a point-of-care rapid test.

LAMPrimers iQ: New primer design software for loop-mediated isothermal amplification (LAMP)

Nucleic acids amplification is a widely used technique utilized for different manipulations with DNA and RNA. Although, polymerase chain reaction (PCR) remains the most popular amplification method, isothermal approaches are gained more attention last decades. Among these, loop-mediated isothermal amplification (LAMP) became an excellent alternative to PCR. LAMP requires an increased number of primers and, therefore, is considered a highly specific amplification reaction compared to PCR. LAMP primers design is still a non-trivial task, and all niceties should be taken into account during their selection. Here, we report on a new program called LAMPrimers iQ destined for high-quality LAMP primers design. LAMPrimers iQ is based on an original algorithm considering rigorous criteria for primers selection. Unlike alternative programs, LAMPrimers iQ can process long DNA or RNA sequences, and completely avoid primers that can form homo- and heterodimers. The quality of the primers designed was checked using SARS-CoV-2 coronavirus RNA as a model target. It was shown that primers selected with LAMPrimers iQ provide higher specificity and reliable detection of viral RNA compared to those obtained by alternative programs. The program is available at https://github.com/Restily/LAMPrimers-iQ.

Comparative genomic analysis of Mycoplasma related to cell culture for infB gene-based loop-mediated isothermal amplification.

Mycoplasma contamination in cell culture affects the properties of cell lines. Gold standard detection by microbiological culture takes days and requires specialists. The polymerase chain reaction and loop-mediated isothermal amplification (LAMP) are fast molecular options, but LAMP only requires one heating block for DNA amplification. This study presents a comparative genomic analysis of Mycoplasma species to identify common target genes different from the rrsA gene, which encodes 16S rRNA. The aim is to implement a LAMP assay to detect Mycoplasma species, reducing the time and specialized equipment required for detection. We performed a comparative genomic analysis through Mauve software and the GView server and selected infB and clpB genes as target candidates for designing LAMP primers. We evaluated both genes by multiple sequence alignment (MSA). The infB gene presented the best score MSA assessment with lower odd-log values (5,480,281) than other genes. We selected the infB gene to design LAMP primers specific to Mycoplasma spp. We used these primers to implement LAMP at 63°C for 30min, which showed 100% positive amplifications for detecting Mycoplasma spp. In conclusion, we present a methodology utilizing the infB gene-based LAMP assay to detect three of the six most prevalent Mycoplasma species in cell culture.

Comparison of loop-mediated isothermal amplification, multiplex PCR, and REP- PCR techniques for identification of carbapenem-resistant Acinetobacter baumannii clinical isolates

Background and Objectives: Acinetobacter baumannii, an opportunistic pathogen, is related to hospital-acquired infections and increased mortality. This study aimed to develop the loop-mediated isothermal amplification (LAMP) test for the fast-de- tecting of A. baumannii isolates as well as determining genetic relatedness for these isolates via the REP-PCR technique.
 Materials and Methods: LAMP primers and multiplex PCR primers were designed for recognizing A. baumannii isolates harboring the bla SHV-1 , bla PER-1 , bla , AMPC, qnr, and aac (6)-1 genes, were collected (October 2020 to February 2021) from Shahid Motahari Hospital, Tehran, Iran. Combination disc test (CDT) results were used to assess the phenotypic iden- tification of isolates from ESBL producers. The sensitivity of the LAMP method was evaluated using a range of serial dilu- tions of genomic DNA. Results were compared between the LAMP technique, and multiplex PCR. The genetic diversity of clinical isolates was determined by REP-PCR.
 Results: Among one hundred A. baumannii samples and based on the combined disc test, 56% of isolates were ESBL pro- ducers. The sensitivity of the LAMP technique for the identification of A. baumannii was 4.06 ng/μl whilst the multiplex PCR was (16.2 ng/μl). Regarding multiplex PCR, (68%) of the isolates were bla SHV-1 positive, (40%) bla , (85%) aac (6́)-1, AMPC (67%), bla TEM-1 (63%), and (15%) qnr respectively. While in LAMP, (69%) of isolates were bla positive, (86%) aac (6')-1, and (20%) qnr. The results of AMPC, bla TEM-1 , and bla PER-1 genes showed 100% compatibility between multiplex PCR and LAMP assays. The results of REP-PCR indicated there were 17 clones, clone A at 14% was the most prevalent of the isolates.
 Conclusion: Wherever equipment and financial constraints are crucial, the LAMP test offers a better and more potent detec- tion rate for the identification of A. baumannii isolates than multiplex PCR. Furthermore, the genetic diversity of A. bauman- nii in these clinical isolates showed frequent commonality of genotypes.

LAMPPrimerBank, a manually curated database of experimentally validated loop-mediated isothermal amplification primers for detection of respiratory pathogens.

The emergence of coronavirus disease 2019 (COVID-19) has once again affirmed the significant threat of respiratory infections to global public health and the utmost importance of prompt diagnosis in managing and mitigating any pandemic. The nucleic acid amplification test (NAAT) is the primary detection method for most pathogens. Loop-mediated isothermal amplification (LAMP) is a rapid, simple, sensitive, and specific epitome of isothermal NAAT performed using a set of four to six primers. Primer design is a fundamental step in LAMP assays, with several complexities and experimental screening requirements. To address this challenge, an online database is presented here. Its workflow comprises three steps: literature aggregation, data curation, and database and website implementation. LAMPPrimerBank ( https://lampprimerbank.mathematik.uni-marburg.de ) is a manually curated database dedicated to experimentally validated LAMP primers, their peculiarities of assays, and accompanying literature, with a primary emphasis on respiratory pathogens. LAMPPrimerBank, with its user-friendly web interface and an open application programming interface, enables the accelerated and facile exploration, comparison, and exportation of LAMP primer sequences and their respective information from the massively scattered literature. LAMPPrimerBank currently comprises LAMP primers for diagnosing viral, bacterial, and fungal respiratory pathogens. Additionally, to address the challenge of false-positive results generated by nonspecific amplifications, LAMPPrimerBank computationally predicted and visualized the sizes of LAMP products for recorded primer sets in the database. LAMPPrimerBank, as a pioneering database in the rapidly expanding field of isothermal NAAT, endeavors to confront the two challenges of the LAMP: primer design and discrimination of false-positive results.

Development of RT-qPCR and loop-mediated isothermal amplification (LAMP) assays for the rapid detection of Rahnella aquatilis in fish

Rahnella aquatilis is widely recognized as an opportunistic pathogen of fish, but molecular detection and diagnostic techniques for it remain to be developed. In a previous study, we isolated the pathogenic R. aquatilis strain KCL-5 from diseased crucian carp Carassius auratus and identified it as a new fish pathogen causing enteritis and septicemia. Here, we describe novel real-time quantitative PCR (RT-qPCR) and loop-mediated isothermal amplification (LAMP) assays for the rapid and sensitive detection of R. aquatilis. Specific RT-qPCR and LAMP primers were developed for the outer membrane protein (OmpA) gene. Their reaction systems were optimized using the recombinant plasmid pMD18-T containing the OmpA gene and their specificities were confirmed using various bacterial fish pathogens including Aeromonas hydrophila, A. veronii, A. allosaccharophila, Yersinia ruckeri, Vibrio vulnificus, V. alfacsensis, Providencia rettgeri, and R. aquatilis. The sensitivities of the RT-qPCR and LAMP assays were further evaluated using serial dilutions of the positive recombinant plasmid pMD18-T/OmpA. Both the RT-qPCR and LAMP assays were able to reliably detect the presence of R. aquatilis with high accuracy and a specificity of 100%. The lowest detection limits of the RT-qPCR and LAMP assays were 2.3 copies/μL and 2.3 × 101 copies/μL, respectively. To the best of our knowledge, this is the first report of RT-qPCR and LAMP methods for R. aquatilis detection, and provides a means for its rapid detection and diagnosis in fish.

Rapid Visual LAMP Method for Detection of Genetically Modified Organisms.

We developed a novel loop-mediated isothermal amplification (LAMP) method using DNA captured on polyacrylamide microparticles (PAMMPs) as templates (PAMMPs@DNA-LAMP) for rapid qualitative detection of genetically modified organisms (GMOs). Here, DNA was extracted by a fast and cost-effective method using PAMMPs. Four LAMP primers were designed for the PAMMPs@DNA-LAMP method to detect the cauliflower mosaic virus 35S (CaMV35S) promotor in GMOs. We thus developed this method for rapid extraction of DNA (5-10 min) and fast amplification of DNA within ∼30 min at a constant temperature of 63 °C. Moreover, the DNA captured by PAMMPs (PAMMPs@DNA) could be effectively detected by both conventional and quantitative PCR (qPCR) and LAMP. The PAMMPs@DNA-LAMP method was validated with high specificity, sensitivity, and performance for practical sample analysis. This assay detected 0.01% target sequences, which had a high specificity like qPCR and better than the conventional PCR (cPCR). Furthermore, PAMMPs@DNA-LAMP was successfully used to extract and detect DNA from food samples of the major crops (soybean, maize, rice, etc.). In summary, a novel PAMMPs@DNA-LAMP assay has been developed, which has higher sensitivity and spends less time than the cPCR detection using the conventional DNA extracted process. This method offers a novel approach for rapid detection of GMOs in the field.

LAMP (Loop-mediated isothermal amplification) assay for rapid identification of Varroa mites

Varroa mites are serious pests of European honeybees (Apis mellifera). For detection of Varroa mite, a new molecular LAMP-based assay has been developed, which retains the body of the mite intact for morphological identification. Six novel Varroa LAMP primers were designed from existing DNA sequences of the COI locus to target V. destructor and V. jacobsoni, providing the ability to tell them apart from other non-target beehive associated mite and insect species. This LAMP assay is specific in detecting these Varroa species and has been tested on specimens originating from multiple countries. It produces amplification of V. destructor and V. jacobsoni in 16 ± 3.4 min with an anneal derivative of 78 ± 0.5 °C whilst another Varroa species,V. underwoodi, showed late amplification. A gBlock gene fragment, used here as a positive control has a different anneal derivative of 80 °C. Three non-destructive DNA extraction methods (HotShot, QuickExtract and Xtract) were tested and found to be suitable for use in the field. The LAMP assay was sensitive to very low levels of Varroa DNA, down to 0.24 picogram (~ 1 × 10 copies/µL of Varroa gBlock). This is a new molecular tool for rapid and accurate detection and identification of Varroa mites for pest management, in areas where these mites do not occur.

Development of extension-mediated self-folding isothermal amplification technology for SARS-CoV-2 diagnosis

The coronavirus disease (COVID-19) pandemic has highlighted the importance of rapid and accurate diagnosis, and loop-mediated isothermal amplification (LAMP) has become a popular method because of its powerful amplification ability using a simple instrument such as a heater or water bath. However, LAMP has limitations such as the complexity of primer design and the difficulty of designing sequence-specific probes, leading to non-specific amplicons and false-positive results.To overcome these limitations, we developed a novel isothermal amplification system called the Extension-mediated self-folding Isothermal amplification Technology (ExIT). ExIT uses a newly designed, self-folding primer (SP) with two key functions. Hairpin structures are formed when the extended strand of the SP hybridizes, exposing the priming site for continuous binding of the new SP. This results in exponential amplification with only two primers, unlike conventional LAMP primer systems. Additionally, an unnatural base was introduced into the SP, which terminated the extension of polymerase and generated a ssDNA amplicon. This makes it easier to design and apply probes, reducing the possibility of false-positive results even if non-specific amplicons are produced.Through this strategy, we confirmed a sensitivity of 90 copies (3.6 copies/μL) and verified the specificity by testing for the presence or absence of non-complementary targets. Therefore, the validation of the ExIT was completed. In conclusion, ExIT will be key to solving the complexity of conventional LAMP design and offers great potential for successfully introducing sequence-specific probes to improve false positives.

Genetic Authentication of the Medicinal Plant Portulaca oleracea Using a Quick, Precise, and Sensitive Isothermal DNA Amplification Assay

Portulaca oleracea (PO) is a commonly known medicinal crop that is an important ingredient for traditional Chinese medicine (TCM) due to its use as a vegetable in the diet. PO has been recorded to be frequently adulterated by other related species in the market of herbal plants, distorting the PO plant identity. Thus, identification of the botanical origin of PO is a crucial step before pharmaceutical or functional food application. In this research, a quick assay named “loop-mediated isothermal amplification (LAMP)” was built for the specific and sensitive authentication of PO DNA. On the basis of the divergences in the internal transcribed spacer 2 (ITS2) sequence between PO and its adulterant species, the LAMP primers were designed and verified their specificity, sensitivity, and application for the PO DNA authentication. The detection limit of the LAMP assay for PO DNA identification specifically was 100 fg under isothermal conditions at 63 °C for 30 min. In addition, different heat-processed PO samples can be applied for use in PO authentication in the LAMP assay. These samples of PO were more susceptible to the effect of steaming in authentication by PCR than boiling and drying treatment. Furthermore, commercial PO samples pursued from herbal markets were used to display their applicability of the developed LAMP analysis for PO postharvest authentication, and the investigation found that approximately 68.4% of PO specimens in the marketplace of herbal remedies were adulterated. In summary, the specific, sensitive, and rapid LAMP assay for PO authentication was first successfully developed herein, and its practical application for the inspection of adulteration in PO samples from the herbal market was shown. This LAMP assay created in this study will be useful to authenticate the botanical origin of PO and its commercial products.

Gold nanostructures modified carbon-based electrode enhanced with methylene blue for point-of-care COVID-19 tests using isothermal amplification

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) and RNA-dependent RNA polymerase (RdRP) genes were detected via electrochemical measurements using a screen-printed carbon electrode (SPCE) (3-electrode system) coupled with a battery-operated thin-film heater based on the loop-mediated isothermal amplification (LAMP) technique. The working electrodes of the SPCE sensor were decorated with synthesized gold nanostars (AuNSs) to obtain a large surface area and improve sensitivity. The LAMP assay was enhanced using a real-time amplification reaction system to detect the optimal target genes (E and RdRP) of SARS-CoV-2. The optimized LAMP assay was performed with diluted concentrations (from 0 to 109 copies) of the target DNA using 30 μM of methylene blue as a redox indicator. Target DNA amplification was conducted for 30 min at a constant temperature using a thin-film heater, and the final amplicon electrical signals were detected based on cyclic voltammetry curves. Our electrochemical LAMP analysis of SARS-CoV-2 clinical samples showed an excellent correlation with the Ct value of real-time reverse transcriptase-polymerase chain reaction, indicating successful validation of results. A linear relationship between the peak current response and the amplified DNA was observed for both genes. The AuNS-decorated SPCE sensor with the optimized LAMP primer enabled accurate analysis of both SARS-CoV-2-positive and -negative clinical samples. Therefore, the developed device is suitable for use as a point-of-care test DNA-based sensor for the diagnosis of SARS-CoV-2.

Triplex-Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Immunoassay for the Simultaneous Detection of Three Pathogens of Porcine Viral Diarrhea Syndrome in Swine.

Porcine epidemic diarrhea virus (PEDV), porcine bocavirus (PBoV), and porcine rotavirus (PoRV) are associated with porcine viral diarrhea. In this study, triplex loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick (LFD) was established for the simultaneous detection of PEDV, PoRV, and PBoV. The PEDV-gp6, PoRV-vp6, and PBoV-vp1 genes were selected to design LAMP primers. The amplification could be carried out at 64 °C using a miniature metal bath within 30 min. The triplex LAMP-LFD assay exhibited no cross-reactions with other porcine pathogens. The limits of detection (LODs) of PEDV, PoRV, and PBoV were 2.40 × 101 copies/μL, 2.89 × 101 copies/μL, and 2.52 × 101 copies/μL, respectively. The consistency between rt-qPCR and the triplex LAMP-LFD was over 99% in field samples testing. In general, the triplex LAMP-LFD assay was suitable for the rapid and simultaneous detection of the three viruses in the field.

Integrated Sensing Chip for Ultrasensitive Label-Free Detection of the Products of Loop-Mediated Isothermal Amplification.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that has been widely used for the detection of pathogens in many organisms. Current LAMP-based sensors usually require the LAMP products to be labeled in order for them to be detected. Here, we present a novel label-free LAMP chip, which consists of a nanopore thin-film sensor embedded inside a LAMP reaction chamber. A fraction of LAMP primers is immobilized on the sensor surface, allowing the LAMP products to be synthesized and bound to the sensor surface via immobilized primers. After the LAMP reaction components are removed from the reaction chamber, the amplified LAMP products bound to the sensor surface give rise to significantly increased transducing signals, which can be measured by a portable optical spectrometer through an optical fiber probe. As a demonstration, we used the LAMP chip to detect the causal agent of late blight, Phytophthora infestans, which is one of the most devastating plant pathogens and poses a major threat to sustainable crop production worldwide. We show that this chip can detect as low as 1 fg/μL of P. infestans DNA in 30 min, which corresponds to an attomolar level of 1.6 × 10-6 attomole/μL and is at least 10 times more sensitive than the currently available methods. This label-free sensing technology holds great promise to open up a new avenue for ultrasensitive, highly specific, rapid, and cost-effective point-of-care diagnostics of plant, animal, human, and foodborne pathogens.

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.

Closed-tube visual detection of Atlantic cod (Gadus morhua) using self-quenched primer coupled with a designed loop-mediated isothermal amplification vessel.

Species adulteration has been widely revealed around the world, and the possible reasons include declining stocks in most source areas of the world, poor transparency in the global supply chain, and difficulty in distinguishing the features of processed products. The present work selected Atlantic cod (Gadus morhua) as a case study, and developed a novel loop-mediated isothermal amplification (LAMP) assay for Atlantic cod authentication, where a self-quenched primer and a newly designed reaction vessel were used to realize the endpoint visual detection of the target-specific products. A novel LAMP primer set was designed for Atlantic cod, and the inner primer BIP was selected to label the self-quenched fluorogenic element. The fluorophore's dequenching only occurred along with LAMP elongation for the target species. No fluorescence could be observed with both single-stranded DNA and partially complementary dsDNA of the non-target species. With the novel reaction vessel, both amplification as well as detection were operated in an enclosed device, and visual differentiation of Atlantic cod, negative control, and false positive generated from primer dimers was achieved. The novel assay has proved its specificity and applicability, and could detect as little as 1 pg of Atlantic cod DNA. Moreover, Atlantic cod adulteration as low as 10% could be detected in haddock (Melanogrammus aeglefinus), and no cross-reactivity was observed. The established assay could be a useful tool to detect mislabeling incidents involving Atlantic cod considering the advantages of speed, simplicity and accuracy. © 2023 Society of Chemical Industry.

Development of detection kit for bad oral bacteria - a NASH factor

Oral and systemic health are linked, with bad Streptococcus mutans (S. mutans) linked to increased risk of intracerebral micro-bleeding and heart disease, and Porphyromonas gingivalis (P. gingivalis) reports of links between periodontal disease and non-alcoholic steatohepatitis (NASH), diabetes, rheumatoid arthritis, Alzheimer's disease, premature birth, and underweight birth. Dr Kentaro Nagamine, Department of Clinical Nutrition, Hiroshima International University, Japan, was involved in the loop-mediated isothermal amplification (LAMP) method and is applying this to oral health, developing primers for LAMP of S. mutans and P. gingivalis and has also developed methods to culture oral bacteria. In one project, he is developing a detection kit for bad S. mutans (cnm gene positive S. mutans). The method he is developing is simple and effective compared to existing methods that tend to be complicated and lengthy. It involves taking a saliva sample, which is then cultured and put into the LAMP reaction solution. Then, the target gene is amplified and gene amplification is seen, this confirms that the bacteria is present. Not only can a result be obtained within two days, but the method also has the ability to amplify multiple types of bacteria. Nagamine is collaborating with a dental clinic that is utilising his method and, in the future, he hopes it can be used for dental examinations in numerous countries and regions, particularly in developing countries.