Validation and optimization of the loop-mediated isothermal amplification (LAMP) technique for rapid detection of wheat stripe mosaic virus, a wheat-infecting pathogen

Background:Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP) worldwide, especially among children and debilitated populations. The present study aimed to investigate a loop-mediated isothermal amplification (LAMP) technique for rapid detection of M. pneumoniae in clinical specimens collected from patients with pneumonia.Methods:Throat swabs were collected from 110 outpatients who suffered from pneumonia. Throat swab samples were obtained from patients referred to the hospital outpatient clinics of Tehran University hospitals, Iran in 2017. The presence of M. pneumoniae in the clinical specimens was evaluated by LAMP, PCR and culture methods. Sensitivity and specificity of the LAMP and PCR assays were also determined.Results:Out of 110 specimens, LAMP assay detected M. pneumoniae in 35 specimens. Detection limit of the LAMP assay was determined to be 33fg /μL or ∼ 40 genome copies/reaction. Moreover, no cross-reaction with genomic DNA from other bacteria was observed. Only 25 specimens were positive by the culture method. The congruence between LAMP assay and culture method was ‘substantial’ (ϰ=0.77). Specificity and sensitivity of LAMP assay were 88.2%, 100% in compare with culture. However, the congruence between LAMP assay and PCR assay was ‘almost perfect’ (ϰ=0.86). Specificity and sensitivity of LAMP assay were 92.5%, 100% in compare with PCR.Conclusion:Overall, the LAMP assay is a rapid and cost-efficient laboratory test in comparison to other methods including PCR and culture. Therefore, the LAMP method can be applied in identification of M. pneumoniae isolates in respiratory specimens.

Candida species cause a wide range of opportunistic infections in humans and animals. The detection of Candida species by conventional diagnosis methods is costly and time consuming. This study was conducted for the first time to evaluate and compare a relatively new molecular assay and the loop-mediated isothermal amplification (LAMP) technique with conventional methods for detection of Candida albicans. In this study, 70 different species of Candida identified by conventional methods were cultured on Sabouraud chloramphenicol agar medium and then the genomic DNA was extracted. The LAMP technique was performed using specific primers targeting the ITS2 gene of C. albicans. The analytical sensitivity and specificity of LAMP were measured using a tenfold serial dilution prepared from extracted DNA from standard C. albicans strain from 1ng to 1fg and the DNA samples of other clinical Candida species and three non-Candida yeast. Out of 70 yeast samples analyzed by LAMP technique, 24 samples (34.3%) were positive for C. albicans. Comparison of the results showed that the CHROMagar Candida and germ tube production methods are quite consistent with the LAMP technique, while the agreement amount between the results of carbohydrate assimilation and chlamydoconidia generation assays and LAMP technique was 98.5% and 72.8%, respectively. The detection limits of the LAMP assay were 10fg of the DNA from the standard C. albicans strain. No amplification was observed in the DNA samples of other yeast species and only the DNA sample of standard C. albicans strain was amplified. Based on the results, it can be concluded that the LAMP method is as specific and precise as common diagnostic methods, but is faster, easier deployable or more sensitive. Therefore, this method can be used as a suitable complementary assay for Candida diagnosis in medical diagnostic laboratories and field conditions.

Perkinsus olseni(P.olseni) is one of the important pathogenic parasites of the shellfish.With the purpose of building a rapid,sensitive,accurate and easy to use detection method for P.olseni,we established a P.olseni loop-mediated isothermal amplification assay(LAMP) based on the internal transcribed spacer(ITS) of Perkinsus olseni 5.8S rDNA sequences.We used the online software Primer Explorer V4(http://primerexplorer.jp/e/) and designed a set of 4 LAMP primers(Perk-FIP,Perk-BIP,Perk-F3 and Perk-B3) of the P.olseni,then we optimized the reaction conditions,mainly about the reaction temperature,magnesium ion concentration of the reaction system and the reaction time.After that,we got the P.olseni 25 μL LAMP reaction system,including: 2.5 μL 10 ThermoPol Reaction Buffer,4 μL dNTPs(10 mmol/L each),5 μL Betaine(5 mol/L),1.6 μL Perk-FIP(25 μmol/L),1.6 μL Perk-BIP(25 μmol/L),1 μL Perk-B3(5 μmol/L),1 μL Perk-F3(5 μmol/L),4 μL MgCl2(25 mmol/L),2.3 μL sterile water,1 μL Bst DNA polymerse(8 000 U/mL) and 1 μL DNA template,the optimal reaction temperature is 64 ℃ and the optimal reaction time is 60 min.In this research,the LAMP products were detected mainly using agarose gel electrophoresis and visual inspection of a color change due to addition of fluorescent dye.Before confirming the minimum threshold of the LAMP,we constructed P.olseni positive plasmid,also based on P.olseni 5.8S rDNA ITS sequences.The result shows that the minimum threshold of the LAMP assay is approximately 30 copies of plasmid DNA.We proved that the developed LAMP method was highly specific for P.olseni,and no cross-reaction was observed with other pathogens,such as Perkinsus marinus(P.marinus),Bonamia exitiosa(B.exitiosa),Ichthyobodo sp.and Acute Viral Necrosis Virus(AVNV).A comparative evaluation of the LAMP and PCR assays using 20 Ruditapes philippinarum(R.philippinarum) samples showed that LAMP is more sensitive and accurate than PCR and the shellfish parasite P.olseni is widely distributed in farming shellfish of North shellfish farming area.Totally,these results indicate that the LAMP method is a kind of simple,sensitive,specific,and reliable technique for the detection of P.olseni.The LAMP technique could be used for the detection of P.olseni in the coastal shellfish farms and laboratories with simple equipment.

Blastocystis spp. is one of the most prevalent intestinal parasites with worldwide distribution. Various diagnostic methods with different sensitivities and specificities have been used to detect Blastocystis in clinical samples. The present study aims to develop and evaluate a LAMP assay to detect Blastocystis spp. in AIDS patients for the first time. In this cross-sectional study, 98 AIDS patients with an average CD4 + T lymphocyte count lower than 150 cells/mm3 participated in the study. The presence of Blastocystis spp. in the stool samples of AIDS patients was examined by parasitology (direct wet mount and concentration assays) and molecular (PCR and LAMP) methods. The 18 SSU rRNA genomic target was used to design the specific primers for the PCR and LAMP assays. The specificity of designed primers for the LAMP assay was evaluated using the sequencing of a conventional PCR product by the external LAMP primers. The data were analyzed using the SPSS software and chi-square test and Fischer's exact tests were used and Cohen's Kappa calculates the agreement of the molecular tests. Associations were tested using odd ratios (OR) and 95% confidence intervals (CI) after adjustments. Out of 98 stool samples from patients with AIDS, 9 (9.18%), 13 (13.26%), and 15 (15.30%) samples were detected positive for Blastocystis spp. by parasitology, PCR, and LAMP techniques, respectively. PCR amplification and subsequent sequencing of the product sequences revealed that the obtained partial sequences were identical to the corresponding 18 SSU rRNA sequences reported in GenBank. The higher positivity rate for Blastocystis spp. among studied AIDS patients by LAMP technique compared to other diagnostic methods showed the higher potential and effectiveness of this relatively new described molecular assay for the detection of Blastocystis spp. in AIDS patients. The accurate and rapid detection of emerging intestinal protozoa such as Blastocystis is of clinical importance for better prevention and timely treatment of the disease, especially in immunocompromised patients. The results obtained for the first time showed that the sensitivity and accuracy of the LAMP technique in the diagnosis of Blastocystis spp. in AIDS patients is very high.

Background Neisseria meningitidis (Nm) is a leading causative agent of bacterial meningitis in humans. Traditionally, meningococcal meningitis has been diagnosed by bacterial culture. However, isolation of bacteria from patients’ cerebrospinal fluid (CSF) is time consuming and sometimes yields negative results. Recently, polymerase chain reaction (PCR)-based diagnostic methods of detecting Nm have been considered the gold standard because of their superior sensitivity and specificity compared with culture. In this study, we developed a loop-mediated isothermal amplification (LAMP) method and evaluated its ability to detect Nm in cerebrospinal fluid (CSF).Methodology/Principal FindingsWe developed a meningococcal LAMP assay (Nm LAMP) that targets the ctrA gene. The primer specificity was validated using 16 strains of N. meningitidis (serogroup A, B, C, D, 29-E, W-135, X, Y, and Z) and 19 non-N. meningitidis species. Within 60 min, the Nm LAMP detected down to ten copies per reaction with sensitivity 1000-fold more than that of conventional PCR. The LAMP assays were evaluated using a set of 1574 randomly selected CSF specimens from children with suspected meningitis collected between 1998 and 2002 in Vietnam, China, and Korea. The LAMP method was shown to be more sensitive than PCR methods for CSF samples (31 CSF samples were positive by LAMP vs. 25 by PCR). The detection rate of the LAMP method was substantially higher than that of the PCR method. In a comparative analysis of the PCR and LAMP assays, the clinical sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP assay were 100%, 99.6%, 80.6%, and 100%, respectively.Conclusions/SignificanceCompared to PCR, LAMP detected Nm with higher analytical and clinical sensitivity. This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.

A loop-mediated isothermal amplification (LAMP) technique was employed to develop a simple and rapid method for the detection of tomato leaf curl Bangalore virus (ToLCBaV) in diseased plants of tomato (Solanum lycopersicum). Six sets of primers were designed for LAMP technique targeting the conserved AC1 region and successfully detected ToLCBaV. No reaction was detected in the tissues of healthy plants by either the LAMP or the polymerase chain reaction (PCR). The LAMP products can be visualized by presence or absence of turbidity and staining (0.2μL for 25μL LAMP product) directly in the tube with nucleic acid stain dye which allowed easy detection. Sensitivity of LAMP assay is 100 times of conventional PCR technique. Although, both the LAMP and the PCR methods were capable of detecting ToLCBaV in infected tissues of tomato, the LAMP method would be more useful than the PCR method for detection of ToLCBaV infection in tomato plants because it is more rapid, simple and accurate method.

At present, there are no effective antiviral treatments available for contagious ecthyma, and rapid diagnosis is therefore critical for effective control of the disease. Recently, the invention of a novel LAMP technique that can rapidly amplify nucleic acids with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic acid-based diagnostic tests and has made on-site diagnosis possible. To establish a flexible loop-mediated isothermal amplification (LAMP) assay for the rapid detection of orf virus, two pairs of primers, including outer primers F3/B3 and inner primers FIP/BIP, were designed to amplify the DNA polymerase gene. Optimal time and temperature conditions for LAMP were found to be 45min and 62°C, respectively. The LAMP assay was shown to be specific, with no cross-reactivity with sheeppox virus, goatpox virus, avian molluscum roup virus or vesicular stomatitis virus. Additionally, the sensitivity of the LAMP method was similar to that of real-time PCR and demonstrated greater sensitivity than a conventional polymerase chain reaction (PCR) assay. To assess the utility of LAMP in the detection of orf virus in clinical samples, a total of 35 samples collected from orf virus-infected sheep and goats were tested using the optimized LAMP assay, real-time PCR, and conventional PCR. Of the samples, 26 were found to be positive by LAMP, and 25 (74.3%) were positive by real-time PCR, whereas only 18 (51.4%) were positive by conventional PCR. Our results have shown that the LAMP assay developed in this study can be used for the rapid detection of orf virus.

Objective To investigate the value of loop-mediated isothermal amplification (LAMP) assay in the etiology diagnosis of bacterial pnuemonia through nucleic acid detection of common pathogens in pneumonia patients by LAMP method. Methods Sputum DNA was extracted from 75 pneumonia patients. DNA was amplified by LAMP. The fluorescence signals of products were detected by real-time PCR. The quantitative results were qualitatively analyzed at different cutoff values, and the data were compared with the results of sputum culture. Results The positive ratio of amplified products with LAMP assay at the cutoff value of 1 × 104 was 68.0% ,and the coincidence rate between LAMP assay and sputum culture was 56.0%. The positive ratio of amplified products with LAMP assay at the cutoff value of 1× 105 was 50.7%, and the coincidence rate between LAMP assay and sputum culture was 58.7%.The positive ratio of amplified products with LAMP assay at the cutoff value of 1 × 106 was 30.7%, and the coincidence rate between LAMP assay and sputum culture was 53.3%. There was no statistical significance on the positive rate between sputum culture and LAMP results at the cutoff value of 1 × 105.The detection rate of parts of bacteria by LAMP assay is higher than that by sputum culture at the cutoff value of 1 × 105 ,especially for the harsh bacteria. Conclusions DNA of common pathogens in sputum of patients with bacterial pneumonia can be easily and quickly amplified by LAMP method to identify pathogenic bacteria types. Compared with sputum culture, the bacterial detection rate is higher by LAMP assay at the cutoff value of 1 × 105. Especially for the harsh bacteria, LAMP method has significant advantages. Key words: Bacterial pneumonia; Diagnosis; Loop-mediated isothermal amplification

Staphylococcus pseudintermedius is a global animal pathogen. Traditional identification methods are time-consuming necessitating a more efficient approach. This study validated and enhanced the loop-mediated isothermal amplification (LAMP) technique by integration it with a lateral flow dipstick (LFD) assay for the detection of S. pseudintermedius and methicillin-resistant S. pseudintermedius (MRSP) strains. Conventional identification methods were compared with LAMP and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The isolates were tested for MRSP detection using oxacillin and cefoxitin disk diffusion tests alongside the LAMP assay targeting the mecA gene, a marker for methicillin resistance. Results showed that LAMP combined with LFD effectively detected S. pseudintermedius and MRSP. This study identified 53 isolates as S. pseudintermedius by conventional and LAMP methods, with MALDI-TOF MS correctly identifying 39.62% (21/53). The mecA gene, crucial for methicillin resistance, was detected in all PCR-positive isolates (n = 33) by LAMP, while the disk diffusion method identified 69.70% (23/33) of mecA-positive strains. LAMP and conventional methods exhibited superior accuracy, sensitivity, and specificity (all 100%) compared to MALDI-TOF MS, which showed lower sensitivity (39.62%) for S. pseudintermedius identification. Similarly, the LAMP assay demonstrated higher accuracy, sensitivity, and specificity (all 100%) for MRSP detection compared to the disk diffusion method (83.33%, 69.70%, and 94.87%, respectively). The LAMP assay coupled with the LFD method proved suitable for routine bacterial identification in laboratories, offering adequate sensitivity and specificity with simple steps and short reaction time.

Rapid diagnosis of lymph node metastasis in lung cancer with loop-mediated isothermal amplification assay using carcinoembryonic antigen–mRNA

The purpose of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the rapid, sensitive detection of Vibrio harveyi in mariculture shellfish. A set of four primers, two outer and two inner primers, were designed from the toxR gene sequence of V. harveyi. The LAMP reaction was conducted at 65 degrees C for 60 min. There were no cross-reactions with other bacterial strains indicating a high specificity of the LAMP. The detection sensitivity of the LAMP assay for V. harveyi with both of pure cultures and added shellfish cultures is about 10(-5) dilution level (equivalent to 17.2 cells per reaction). The amplification products were detected by visual inspection using SYBR Green I. The detection sensitivity using the LAMP method was 10 times higher than that of conventional PCR. The LAMP assay established in this study is an extremely specific, sensitive and rapid for identification of V. harveyi in mariculture shellfish. This LAMP technique provides an important detecting tool for the detection of V. harveyi infection both in the laboratory and field. This technique is recommended as an applied protocol for health management programme and disease surveillance of in hatcheries as well as in grow-out pond, to prevent the disease outbreak.

Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop-Mediated Isothermal Amplification Reaction

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

Rapid detection of fish pathogens is mandatory for applying the crucial preventive and control measures to reduce fish losses and, consequently, minimize the economic impact of diseases on the fish farm owners. The currently used molecular diagnostic tools of fish infectious agents, such as PCR and RT-PCR, are sensitive and specific but still have some drawbacks. These tools are usually time consuming and laborious, need skilled persons, and require sophisticated devices to be performed. Therefore, next-generation tools for rapid diagnosis of fish infectious diseases were developed to conquer these shortages. One of these novel tools is the loop-mediated isothermal amplification (LAMP) technique. LAMP is considered a more advantageous tool than PCR because it needs only a heating block or a thermostatically controlled water bath as a source of constant temperature. It is considered to be more specific than the PCR assay as it uses 4-6 primers, which may diminish the occurrence of false-positive results. The time required for the amplification process by LAMP is ranging from 30 min to 1 h comparing to 3-5 h in the case of PCR. The visual detection methods coupled with the LAMP assay eliminates the post-run processing for detection of the amplification products. Its sensitivity is either comparable with the PCR or better than it. A variety of LAMP assays were developed for simple and rapid detection of a diversity of fish pathogens. Herein, we describe how to perform a LAMP assay and troubleshoot any potential problem arising during the process.

In this study, a loop-mediated isothermal amplification (LAMP) assay has been developed and evaluated for the rapid and sensitive detection of Verticillium dahliae Kleb., the causal agent of vascular wilts in many economically important crops. LAMP primers were designed based on a previously described RAPD marker, and the LAMP assay was applied for direct detection of V. dahliae grown on medium and from soil samples without DNA purification steps (direct-LAMP). Thirty-two agricultural soil samples from various olive orchards were collected, and the presence of pathogen was detected by LAMP, direct-LAMP and nested-PCR methods. The LAMP methodology could successfully detect V. dahliae with high specificity, and cross-reaction was not observed with different pathogenic and nonpathogenic fungi and bacteria. The LAMP assay was capable of detecting a minimum of 500 and 50 fg of purified target DNA per reaction of V. dahliae ND and D pathotypes, respectively. In contrast, nested-PCR could only detect 5 pg reaction(-1) for both pathotypes. In artificially infested soil samples, the LAMP method detected 5 microsclerotia per gram of soil. Conversely, nested-PCR assay detected 50 microsclerotia g(-1) soil. The detection ratios of LAMP and direct-LAMP protocols were better (26 and 24 positive samples out of 32 agricultural soils analysed, respectively) than that obtained for nested-PCR method (22 positive results). Moreover, direct-LAMP yielded positive detection of V. dahliae in agricultural soil samples within 60-80 min. The newly developed LAMP method was proved to be an effective, simple and rapid method to detect V. dahliae without the need for either expensive equipment or DNA purification. This technique can be considered as an excellent standard alternative to plating and nested-PCR assays for the early, sensitive and low-cost detection of V. dahliae as well as other soilborne pathogens in the field.