Use Of Loop-mediated Isothermal Amplification Research Articles

Salmonella detection with LAMP and qPCR and identification of serovars of interest by multiplex qPCR in poultry carcasses.

Salmonella is present in the poultry production chain and is a major challenge in terms of food safety and animal health. The early Salmonella detection is one of the main tools to control and prevent the transmission of this pathogen. Microbiological isolation and serotyping to identify and differentiate Salmonella serovars are laborious processes, time-consuming, and expensive. Therefore, molecular diagnostic methods can be rapid and efficient alternatives to the detection of this pathogen. Thus, the aim herein was to standardize and evaluate the use of loop-mediated isothermal amplification (LAMP) in comparison with real-time PCR (qPCR) for detection of Salmonella associated with a multiplex qPCR for simultaneous identification and differentiation of S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Gallinarum. The LAMP, qPCR, and multiplex qPCR assays were comparable in specificity. The three techniques were evaluated for specificity for 16 different serovars of Salmonella and for 37 strains of the serovars of interest. The limit of detection and the efficiency of the LAMP, qPCR, and multiplex qPCR reactions were determined. The techniques were applied to 33 samples of chicken carcasses and compared to the results of conventional microbiology for validation. As results, LAMP was specific in the detection of different Salmonella serovars but presented lower limit of detection ranging from 101 to 104 CFU/reaction. In comparison, qPCR could detect less cells (100 to 102 CFU/reaction), reaching equal specificity and better repeatability in the assays. The qPCR multiplexing for identification of the different serovars also showed good specificity, with the detection threshold between entre 101 and 102 CFU/reaction. The results obtained in the analyses on poultry carcasses suggested a correspondence between the results obtained in molecular methods and in conventional microbiology. Thus, the proposed assays are promising for the diagnosis of Salmonella in poultry carcasses, already proved to be faster and more efficient than conventional diagnostics techniques, being of great interest for poultry production, animal, and public health.

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

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

In-the-Field Authentication of Grapevine (Vitis vinifera L.) cv: Albariño Using Chlorotype Discrimination and a Single SNP Interrogation by LAMP

Authentication of grapevine cultivars, Vitis vinifera L., is difficult, especially when analytical specimens lack diagnostic ampelographic characters, which prevents the verification of traceability systems aimed at guaranteeing varietal integrity. This issue is problematic when viticultural strategies and regulations associated with high-value wine-producing geographical areas rely on extensive control and monitoring of valuable cultivars. Varietal identification based on grapevine molecular markers is a standardized methodology that requires a specialised laboratory for its application. In contrast, the use of loop-mediated isothermal amplification (LAMP) allows DNA markers to be characterized quickly and easily, without the need for skilled personnel, allowing implementation in-situ or in-the-field. Simultaneous identification of the chlorotype and the interrogation of a single SNP using a portable device have allowed the first discrimination in-the-field of grafted grapevines, without appreciable ampelographic characters, as belonging to the valuable “Albariño” cultivar. This methodology constitutes a valuable tool for cultivar discrimination and can be efficiently implemented in the traceability of valuable grapevine genetic resources.

Performance of Loop-Mediated Isothermal Amplification (LAMP) Targeting the Nucleocapsid (N) Gene of SARS-CoV-2 for Rapid Diagnosis of COVID-19: Systematic Review and Meta-Analysis

SARS-CoV-2 emerged as a new respiratory virus spreading rapidly to all areas of the world. A systematic review with meta-analysis concerning the use of Loop-Mediated Isothermal Amplification (LAMP) methodology targeting the SARS-CoV-2 nucleoprotein (N) gene was conducted. The search resulted in 229 articles, of which 19 articles were selected to compose the final review. In general, LAMP showed a high specificity in the detection of SARS-CoV-2 and a wide variation of sensitivity values. The LAMP method was considered a fast and highly specific method for SARS-CoV-2 detection; however, some variables may affect its sensitivity and overall performance.

Evaluation of the Clinical Usefulness of Loop-mediated Isothermal Amplification (LAMP) Assay for the Detection of <i>Chlamydia pneumoniae</i>

Evaluation of the Clinical Usefulness of Loop-mediated Isothermal Amplification (LAMP) Assay for the Detection of <i>Chlamydia pneumoniae</i>

Molecular tools in monitoring the outset of tomato leaf curl virus (ToLCV) Transmission in the Philippines

The study validated the use of Loop-Mediated Isothermal Amplification (LAMP) and polymerase chain reaction (PCR) in tracking the possible cause of transmission of the tomato leaf curl virus (ToLCV). Detection of ToLCV was performed by assessing the detection rate from the parallel tests of these molecular methods against the greenhouse-controlled conventional method. Assessment of symptomatic and asymptomatic plants was conducted before sampling and processing for the assays. Plant viruses are commonly transmitted by vectors, most significantly by the Silverleaf whiteflies or Bemisia tabaci. Here, PCR and LAMP detected the virus in seedlings 0-dpi (0-day post-inoculation) even before the symptoms appeared while yellowing and minor curling of leaflet ends were observed in tomato plants at 12 dpi. Results revealed that the transmission of ToLCV occurs not only via the insect vector but this virus can also be transmitted through contaminated seeds and can be expected in soil particles. The results suggest the benefits of using these molecular diagnostic methods for ToLCV in detecting the early stage of infection when symptoms are not yet fully expressed thus, saving the assay time, resources and most importantly, the crops.

Diagnostic Usefulness of Loop-mediated Isothermal Amplification for Intrafamilial Pertussis Infection

Diagnostic Usefulness of Loop-mediated Isothermal Amplification for Intrafamilial Pertussis Infection

Use of Loop-Mediated Isothermal Amplification in a Resource-Saving Strategy for Primary Malaria Screening in a Non-Endemic Setting.

.Malaria is traditionally diagnosed by blood smear microscopy, which requires continuous resource-demanding training. In areas with only a few cases of malaria, a simple and rapid test that can reliably exclude malaria could significantly reduce the need for microscopy and training. We evaluated whether loop-mediated isothermal amplification (LAMP) for screening malaria parasites could reduce the workload in the diagnosis of malaria. Loop-mediated isothermal amplification was used to analyze 38 ethylene-diamine-tetraacetic acid (EDTA) blood samples from 23 patients who had previously been tested for malaria by microscopy, antigen-based rapid diagnostic test (antigen-RDT), and in-house real-time polymerase chain reaction (RT-PCR). The samples included blood with low-level parasitaemia and samples with discrepancies between the results of the different methods. Loop-mediated isothermal amplification detected malaria parasites in 27 of 28 samples that were positive according to in-house RT-PCR. There were negative microscopy results in 10 of these and negative antigen-RDT results in 11. The sample with a negative LAMP result and positive in-house RT-PCR result was from a patient who had recently been treated for low-level Plasmodium falciparum malaria parasitaemia. We found LAMP to be reliable for malaria screening and suitable for replacing microscopy without loss of performance. The low number of LAMP-positive samples needing microscopy can be handled by a limited number of trained microscopists. The time saved on training and documentation was estimated to be 520 working hours yearly in our laboratory. Using LAMP for primary screening of patient samples, we have made a diagnostic workflow that ensures more reliable, faster, and less resource-demanding diagnosis of malaria.

Evaluation of the 3M™ Molecular Detection Assay (MDA) 2 - Cronobacter for the Detection of Cronobacter species in Select Foods and Environmental Surfaces: Collaborative Study, First Action 2018.01.

The 3M™ Molecular Detection Assay (MDA) 2 - Cronobacter combines the use of loop-mediated isothermal amplification to rapidly amplify nucleic acid sequences while using bioluminescence to detect the amplification. Using a paired study design, the MDA 2 - Cronobacter was compared with ISO 22964:2017 for the detection of Cronobacter species in powdered infant formula containing probiotics. Technicians from 11 laboratories from the United States, Mexico, and Croatia participated. Collaborators received test portions with three levels of contamination. Statistical analysis was conducted according to the probability of detection (POD) statistical model. Results obtained for the low-inoculum level test portions produced a difference in POD values obtained from combining all valid collaborator POD data values with 95% confidence intervals of -0.01, -0.12, and 0.10, indicating that the difference between methods was not statistically significant at the 0.05 probability level.

Loop-Mediated Isothermal Amplification on Crude DNA as a Point-of-Care Test for the Diagnosis of Mycoplasma-Related Vaginitis During Early Pregnancy.

Mycoplasma-related vaginitis gradually has been growing as a threat in adults-genitourinary infection contributes to funisitis, spontaneous abortion, and low birth weight. Until now, use of loop-mediated isothermal amplification (LAMP) to detect Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), or Mycoplasma genitalium (MG) has been reported by some researchers. However, previous studies focused on purified DNA as the template for LAMP assay, which is usually extracted via commercial kit. We developed a LAMP assay for rapid detection of UU, MH, and MG genital mycoplasmas using a simple boiling method for DNA extraction, in a cohort of pregnant women with mycoplasma-related vaginitis. We monitored amplicons with the naked eye using SYBR Green I. The cohort in our study showed a prevalence of 22.6% in pregnant women, as detected by UU-LAMP assay. Compared to the polymerase chain reaction (PCR) test with purified DNA, the sensitivity of the UU-LAMP in clinical specimens with crude DNA was 87.5% (95% confidence interval [CI], 64.6%->99.9). For crude DNA specimens, UU-LAMP was more sensitive and reliable than PCR, with a higher agreement rate (96.8%) and Youden index value (0.88). As a point-of-care test, LAMP is a useful, specific, and efficient way to detect genital mycoplasmas in resource-limited settings, especially for crude DNA.

Application of loop-mediated isothermal amplification assay in the detection of herpesvirus of turkey (FC 126 strain) from chicken samples in Nigeria.

Aim:This study was designed to optimize and apply the use of loop-mediated isothermal amplification (LAMP) as an alternative to conventional polymerase chain reaction (PCR) for the detection of herpesvirus of turkeys (HVT) (FC 126 strain) in vaccinated and non-vaccinated poultry in Nigeria.Materials and Methods:HVT positive control (vaccine) was used for optimization of LAMP using six primers that target the HVT070 gene sequence of the virus. These primers can differentiate HVT, a Marek’s disease virus (MDV) serotype 3 from MDV serotypes 1 and 2. Samples were collected from clinical cases of Marek’s disease (MD) in chickens, processed and subjected to LAMP and PCR.Results:LAMP assay for HVT was optimized. HVT was detected in 60% (3/5) and 100% (5/5) of the samples analyzed by PCR and LAMP, respectively. HVT was detected in the feathers, liver, skin, and spleen with average DNA purity of 3.05-4.52 μg DNA/mg (A260/A280) using LAMP. Conventional PCR detected HVT in two vaccinated and one unvaccinated chicken samples, while LAMP detected HVT in two vaccinated and three unvaccinated corresponding chicken samples. However, LAMP was a faster and simpler technique to carry out than PCR.Conclusion:LAMP assay for the detection of HVT was optimized. LAMP and PCR detected HVT in clinical samples collected. LAMP assay can be a very good alternative to PCR for detection of HVT and other viruses. This is the first report of the use of LAMP for the detection of viruses of veterinary importance in Nigeria. LAMP should be optimized as a diagnostic and research tool for investigation of poultry diseases such as MD in Nigeria.

A Rapid and Simple Loop-Mediated Isothermal Amplification Assay for the Detection of Pseudomonas aeruginosa From Equine Genital Swabs

Pseudomonas aeruginosa is a causative agent of infectious reproductive disease in horses. Conventional methods for identification of this organism from equine genital swabs can be time consuming, laborious, and may rely on the presence of live bacteria. However, the use of loop-mediated isothermal amplification (LAMP) provides a simple and rapid detection method. We aimed to develop and evaluate a simple-to-use and interpret LAMP assay, based on the P. aeruginosa ecfX gene, capable of detecting P. aeruginosa from DNA extracted directly from equine genital swabs without culture or prior enrichment. The specificity of the assay was determined using DNA extracted from 29 different bacterial species, as well as in silico. The diagnostic sensitivity of the assay was determined through analysis of DNA extracted directly from 46 equine genital swabs. A P. aeruginosa–specific LAMP assay that could detect as few as 11 copies of its target in less than 15 minutes was developed. The reaction amplicon could be detected by real-time fluorescence or using a simple lateral flow device. The assay was specific to its target, showing no cross-reactivity or interference when tested against DNA extracted from 29 other bacterial species, as well as in silico. The diagnostic sensitivity of the assay was 86.8% (39 of 46) and 88.5% (40 of 46), when the amplicon was detected by real-time fluorescence and lateral flow device technology, respectively. The P. aeruginosa–specific LAMP assay provides a simple and rapid means of detecting the presence of viable and nonviable P. aeruginosa directly from equine genital swabs.

Rapid Visual Detection of Highly Pathogenic Streptococcus suis Serotype 2 Isolates by Use of Loop-Mediated Isothermal Amplification

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen that causes considerable economic losses to the pig industry and significantly threatens public health worldwide. The highly pathogenic S. suis 2, which contains the 89K pathogenicity island (PAI), has caused large-scale outbreaks of infections in humans, resulting in high mortality rates. In this study, we established two loop-mediated isothermal amplification (LAMP)-based assays that can rapidly detect S. suis 2 and the 89K PAI and can be performed simultaneously under the same conditions. Further, based on the findings of these two LAMP assays and using the same set of serially diluted DNA samples, we compared the sensitivities of different LAMP product detection methods, including SYBR green detection, gel electrophoresis, turbidimetry, calcein assays, and hydroxynaphthol blue detection. The results suggest that target genes can be amplified and detected within 48 min under 63°C isothermal conditions. The sensitivity of tests for S. suis 2 detection varies between detection methods and reaction systems, indicating that for each LAMP reaction system, multiple detection methods should be performed to select the optimal one. The sensitivities of the optimized methods (7.16 copies/reaction) in the present study were identical to those of the real-time PCR assay, and the test results for reference strains and clinical samples showed that these LAMP systems have high specificities. Thus, since the LAMP systems established in this study are simple, fast, and sensitive, they may have good clinical potential for detecting the highly pathogenic S. suis 2.

Improved loop-mediated isothermal amplification for HLA-DRB1 genotyping using RecA and a restriction enzyme for enhanced amplification specificity

Our aim was to test and develop the use of loop-mediated isothermal amplification (LAMP) for HLA-DRB1 genotyping. Initially, we found that the conventional LAMP protocols produced non-specific and variable amplification results depending on the sample DNA conditions. Experiments with different concentrations of DNase in the reaction mixture with and without T4 DNA ligase-treated samples suggested that the strand displacement activity of DNA polymerase in LAMP, at least in part, started from randomly existing nicks because T4 DNA ligase treatment of sample DNA resulted in no amplification. Such non-specific amplification due to the randomly existing nicks was improved specifically by the addition of RecA of Escherichia coli and a restriction enzyme, for example, PvuII, to the reaction mixture. We applied the modified LAMP (mLAMP) (1) to detect specific HLA-DRB1 alleles by using only specific primers for amplification or (2) for genotyping in multiple samples with a multi-probe typing system. In the latter case, HLA-DRB1 genotyping was developed by combining the mLAMP with amplicon capture using polymorphic region-specific probes fixed onto the bottom of the wells of a 96-well plate and the captured amplicons visualized as a black spot at the bottom of the well. The multi-probe human leukocyte antigen (HLA) typing method and the specific HLA allele detection method could be applied for point-of-care testing due to no requirement for specific and expensive instruments.

Use of loop-mediated isothermal amplification for detection of Ophiostoma clavatum, the primary blue stain fungus associated with Ips acuminatus.

Loop-mediated isothermal amplification (LAMP) is an alternative amplification technology which is highly sensitive and less time-consuming than conventional PCR-based methods. Three LAMP assays were developed, two for detection of species of symbiotic blue stain fungi associated with Ips acuminatus, a bark beetle infesting Scots pine (Pinus sylvestris), and an additional assay specific to I. acuminatus itself for use as a control. In common with most bark beetles, I. acuminatus is associated with phytopathogenic blue stain fungi involved in the process of exhausting tree defenses, which is a necessary step for the colonization of the plant by the insect. However, the identity of the main blue stain fungus vectored by I. acuminatus was still uncertain, as well as its frequency of association with I. acuminatus under outbreak and non-outbreak conditions. In this study, we employed LAMP technology to survey six populations of I. acuminatus sampled from the Southern Alps. Ophiostoma clavatum was detected at all sampling sites, while Ophiostoma brunneo-ciliatum, reported in part of the literature as the main blue stain fungus associated with I. acuminatus, was not detected on any of the samples. These results are consistent with the hypothesis that O. clavatum is the main blue stain fungus associated with I. acuminatus in the Southern Alps. The method developed in the course of this work provides a molecular tool by which it will be easy to screen populations and derive important data regarding the ecology of the species involved.

The use of Loop-mediated Isothermal Amplification (LAMP) to detect the re-emerging Human African Trypanosomiasis (HAT) in the Luangwa and Zambezi valleys

BackgroundLoop-mediated isothermal amplification (LAMP) is a novel strategy which amplifies DNA with high sensitivity and rapidity under isothermal conditions. In the present study, the performance of the repetitive insertion mobile element (RIME)-LAMP and human serum resistance-associated gene (SRA)-LAMP assays were evaluated using clinical specimens obtained from four male patients from Luangwa and Zambezi valleys in Zambia and Zimbabwe, respectively.FindingsThe cases reported in this preliminary communication were all first diagnosed by microscopy, through passive surveillance, and confirmed by both RIME-LAMP and SRA-LAMP. A good correlation between microscopy and LAMP was observed and contributed to staging and successful treatment of patient. RIME-LAMP and SRA-LAMP complimented each other well in all the cases.ConclusionsBoth RIME-LAMP and SRA-LAMP were able to detect Trypanosoma brucei rhodesiense DNA in patient blood and CSF and hence confirmed HAT in the parasitaemic patients. Our study indicates that the LAMP technique is a potential tool for HAT diagnosis, staging and may be useful for making therapeutic decisions. However, no statistically significant conclusion may be drawn due to the limited sample size used in the present study. It is thus imperative to conduct a detailed study to further evaluate the potential of LAMP as a bedside diagnostic test for HAT.

Use of Loop-Mediated Isothermal Amplification of DNA for the Rapid Detection of HIV/AIDS Related Opportunistic Infections (CMV &TB) in Clinical Specimens

This review will describe about the loop mediated isothermal amplification (LAMP) and its advantages in detection of opportunistic infections in HIV infected individuals. HIV infection is the strongest risk factor for development of tuberculosis (TB) and Cytomegalovirus (CMV) conversely both TB and CMV are the commonest HIV-associated opportunistic diseases worldwide. Loop-mediated isothermal amplification (LAMP) can amplify DNA with high specificity, efficiency and rapidity under isothermal condition. One of the great advantages of the LAMP assay is that amplification can be monitored with the naked eye using SYBR green dye. This sort of detection system is easy and helpful in discriminating infectious and non infectious samples. In view of the advantages of rapid amplification, and easy detection, LAMP has potential applications for clinical diagnosis in developing countries without requiring experienced persons and sophisticated equipment.

Use of loop-mediated isothermal amplification of DNA for the rapid detection of Mycobacterium tuberculosis in clinical specimens

Loop-mediated isothermal amplification (LAMP) is a recently developed molecular method that has been successfully implemented in the detection of Mycobacterium tuberculosis in clinical specimens. LAMP has several advantages, such as rapidity, high sensitivity, ease of application and cost-effectiveness. As a result, it is anticipated that its use for the detection of M. tuberculosis is likely to become widespread, especially in low-resource countries. The present review aimed to present this method and all of the available information on its implementation in the detection of M. tuberculosis in clinical specimens.

Sex distinction of asparagus by loop-mediated isothermal amplification and observation of seedling phenotypes

Asparagus (Asparagus officinalis L.) is a dioecious plant. In general, male and female plants are used for open-field culture and intensive cultivation, respectively. Farmers distinguish between the sexes by observing the form of the flower organs. However, because flowering begins 2–3 years after planting, the sexes cannot be differentiated at transplantation by using this method, and planting of an all-male population is not possible. In this study, the usefulness of loop-mediated isothermal amplification (LAMP), a simple method of gene amplification, for sex distinction at the DNA level was determined. In addition, the phenotypic differences in seeds and seedlings of male and female plants were investigated for application as a method of early sex distinction. By using the LAMP method, the sex could be correctly identified in 100% of the seedlings, suggesting that this method is effective for sex distinction at the gene level. Principal component analysis was conducted with 11 selected parameters after investigating the seeds and seedlings of both male and female plants. The results revealed that male plants tend to have many stalks or cladophylls and female plants tend to have large plant forms, suggesting that the sexes can be distinguished by the external appearance of the seedlings before planting. LAMP and observation of the seedling phenotypes could be useful methods of sex distinction for increasing the efficiency of asparagus breeding.

Use of loop-mediated isothermal amplification of the IS900 sequence for rapid detection of cultured Mycobacterium avium subsp. paratuberculosis.

We evaluated the usefulness of loop-mediated isothermal amplification (LAMP) in detecting specific gene sequences of Mycobacterium avium subsp. paratuberculosis (MAP). A total of 102 primer sets for LAMP was designed to amplify the IS900, HspX, and F57 gene sequences of MAP. Using each of two primer sets (P-1 and P-2) derived from the IS900 fragment, it was possible to detect MAP in a manner similar to that used with nested PCR. The sensitivity of LAMP with P-1 was 0.5 pg/tube, which was more sensitive than nested PCR. When P-2 was used, 5 pg/tube could be detected, which was the same level of sensitivity as that for nested PCR. LAMP with P-1 was specific. Although only 2 Mycobacterium scrofulaceum strains out of 43 non-MAP mycobacterial strains were amplified, the amplification reaction for these strains was less efficient than for MAP strains, and their products could be distinguished from MAP products by restriction digestion. LAMP with P-2 resulted in very specific amplification only from MAP, the same result obtained with nested PCR. Our LAMP method was highly specific, and the white turbidity of magnesium pyrophosphate, a by-product of the LAMP reaction, allowed simple visual detection. Our method is rapid, taking only 2 h, compared with 4 h for nested PCR. In addition, the LAMP method is performed under isothermal conditions and no special apparatus is needed, which makes it more economical and practical than nested PCR or real-time PCR. These results indicate that LAMP can provide a rapid yet simple test for the detection of MAP.