Rapid and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification combined with lateral flow dipstick targeted to rpoS gene

A novel loop-mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay was developed and evaluated for the detection of Vibrio vulnificus. Biotinylated LAMP amplicons were produced by a set of six designed primers that recognized the V. vulnificus RNA polymerase subunit sigma factor S (rpoS) gene followed by hybridization with an FITC-labeled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65 °C. The LAMP–LFD method accurately identified 14 isolates of V. vulnificus but did not detect 25 non-vulnificus Vibrio isolates and 37 non-Vibrio isolates. The sensitivity of LAMP–LFD for V. vulnificus detection in pure culture was 1.5 × 10³ CFU ml⁻¹ or equivalent to 2.8 CFU per reaction. In the case of spiked oyster samples without enrichment, the detection limit for V. vulnificus was 1.2 × 10⁴ CFU g⁻¹ or equivalent to 11 CFU per reaction. The results show that this method appears to be accurate, precise and valuable tool for identification of V. vulnificus and can be used efficiently for detection of V. vulnificus in contaminated food sample.     

Development of a loop-mediated isothermal amplification assay for detection of Trichomonas vaginalis

A loop-mediated isothermal amplification (LAMP) assay targeting the 2-kbp repeated DNA species-specific sequence was developed for detection of Trichomonas vaginalis, the causative agent of trichomoniasis. The analytical sensitivity and specificity of the LAMP assay were evaluated using pooled genital swab and urine specimens, respectively, spiked with T. vaginalis trophozoites. Genital secretion and urine did not inhibit the detection of the parasite. The sensitivity of the LAMP was 10–1000 times higher than the PCR performed. The detection limit of LAMP was 1 trichomonad for both spiked genital swab and urine specimens. Also, LAMP did not exhibit cross-reactivity with closely-related trichomonads, Trichomonas tenax and Pentatrichomonas hominis, and other enteric and urogenital microorganisms, Entamoeba histolytica, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. This is the first report of a LAMP assay for the detection of T. vaginalis and has prospective application for rapid diagnosis and control of trichomoniasis.     

Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures

Staphylococcus aureus is the most important pathogen in nosocomial infections, including bloodstream infections. Prompt identification of S. aureus from blood cultures and detection of methicillin resistance are essential in cases of suspected sepsis. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63°C) with high specificity, efficiency, and rapidity, was applied to detect methicillin-resistant S. aureus (MRSA) directly from positive blood culture bottles. MRSA-LAMP, which targets the spa gene, encoding S. aureus-specific protein A, and the mecA gene, encoding penicillin-binding protein-2′ for methicillin resistance, could detect MRSA within 2 h after the blood culture signal became positive. The diagnostic values of LAMP, compared to a duplex real-time polymerase chain reaction (Drt-PCR) assay, were 92.3% and 96.2% sensitivity, 100% and 100% specificity, 100% and 100% positive predictive value (PPV), and 96.9% and 98.4% negative predictive value (NPV), respectively. These two methods had almost the same results, but the LAMP method is more cost-effective and provides excellent availability for rapid examination in a hospital clinical laboratory. Therefore, the LAMP assay appears to be a sensitive and reliable new method to diagnose MRSA bloodstream infection for appropriate antibiotic therapy.     

Application of a loop-mediated isothermal amplification method for the detection of pathogenic Leptospira

Leptospirosis is an emerging infectious disease, which is considered to be the most widespread zoonotic disease in the world. There are more than 230 known serovars in the genus Leptospira. A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of pathogenic Leptospira spp. was developed and evaluated through amplification of the lipL41 gene coding for the outer membrane protein LipL41. The LAMP assay did not rely on the isolation and culture of leptospires, and no cross-reactivity was observed with other bacterial species. A SYBR Green I-based LAMP assay was also carried out for the real-time detection of DNA amplification. The lower detection limit of the LAMP assay was approximately 100 copies, which was the same as the polymerase chain reaction (PCR) and real-time PCR assays. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis of the amplified product. The LAMP assay is easy to perform and inexpensive, and so may be applied in the rapid and specific diagnosis of Leptospira.     

Rapid diagnosis of largemouth bass ranavirus in fish samples using the loop-mediated isothermal amplification method

Largemouth bass ranavirus (LMBV) has been recognized as the causative pathogen responsible for infectious skin ulcerative syndrome in cultured largemouth bass in China. A fast and simple LMBV detection method is urgently needed. Here, a loop-mediated isothermal amplification (LAMP) assay was established for the detection of this virus using primers targeting the major capsid protein gene of LMBV. The amplification conditions were optimized; the assay was specific for the diagnosis of LMBV, as there was no cross-reactivity with other four Iridoviridae viruses (large yellow croaker iridovirus, Singapore grouper iridovirus, tiger frog virus, and soft-shelled turtle iridovirus), grass carp reovirus, white spot syndrome virus, or healthy largemouth bass. The sensitivity of the LAMP assay was found to be 8.55 × 10¹ copies/μL of LMBV DNA, which was 10-fold higher than that of the conventional PCR. Application of the LAMP assay was evaluated using 10 clinical samples, and the results indicated the reliability of the test as a rapid, field diagnostic tool for LMBV detection. Thus, the simplicity and nearly instrument-free LAMP method provides an alternative for rapid and sensitive detection of LMBV and has great potential for early diagnosis of LMBV infection in the farm.     

A rapid,simple, and sensitive loop-mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal swab samples

Loop-mediated isothermal amplification (LAMP) method was designed for clinical diagnosis of Vibrio cholerae carrying the ctxA gene. The detection limits of the method were 5 fg of purified genomic DNA/reaction and 0.54 CFU/reaction. The method was applied to rectal swab samples from cholera patients and healthy volunteers (19 subjects each) and yielded the same results as the “gold standard” culture method, while the polymerase chain reaction-based method failed to detect V. cholerae in 8 of the positive samples. Direct application of this LAMP method without precultivation enabled the rapid detection of 5 asymptomatic carriers from rectal swabs of 21 household contacts of cholera patients. This LAMP method could be a sensitive, specific, inexpensive, and rapid detection tool for V. cholerae carrying the ctxA gene in the clinical laboratory and in the field.     

Loop-mediated isothermal amplification method targets to the phoP gene for detection of Yersinia enterocolitica

A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of Yersinia enterocolitica. The assay used specifically designed primers to target within the phoP gene and correctly identified all 37 strains of Y. enterocolitica and 50 non-Y. enterocolitica strains. The probability of detection was 100%, when the DNA of extracted from 10¹ CFU Y. enterocolitica was used as template in LAMP assay. Prior to the LAMP assay, a sample preparation protocol was applied that included a pre-enrichment step in Luria–Bertani broth, followed by extraction and purification of DNA. In this way, 102 various food samples were investigated for Y. enterocolitica including 79 minced pork samples and 23 powdered milk samples. The accuracy of LAMP was shown to be 100% when compared to the standard method, ISO 10273. This combination of sample enrichment, and LAMP assay can detect 2.2 CFU per 100 g food samples. The overall analysis time for the LAMP assay was approximately 24 h. This is in contrast to 5 days of analysis time required for the traditional culture method. Consequently, the LAMP described here, has the potential to become a standardized method for the rapid detection of Y. enterocolitica in diagnostic laboratories once further validated by inter-laboratory studies.     

Application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post-kala-azar dermal leishmaniasis

Loop-mediated isothermal amplification (LAMP) is at the forefront in the search for innovative diagnostics for rapid and specific amplification of target DNA under isothermal conditions. We have applied LAMP assay using SYBR Green for clear-cut naked eye detection of Leishmania (Leishmania) donovani in 200 clinical samples of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). The assay was positive in 53/55 VL blood samples (sensitivity, 96.4%; 95% confidence interval [CI], 87.7–99%), 15/15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI, 79.6–100%), 60/62 PKDL tissue biopsy samples (sensitivity, 96.8%; 95% CI, 88.9–99.1%), and 1/68 control samples (specificity, 98.5%; 95% CI, 92.1–99.7%). The assay was specific for L. (L.) donovani, the causative species for VL and negative for L. (L.) infantum, L. (L.) tropica, and L. (L.) major. This is the first comprehensive clinical study demonstrating the applicability of the LAMP assay for a rapid and reliable molecular diagnosis of VL and PKDL.     

Development of a PCR- and probe-based test for the sensitive and specific detection of the Dog Heartworm,Dirofilaria immitis , in its mosquito intermediate host

The mosquito-borne filarial worm, Dirofilaria immitis, causes heartworm disease in dogs. Detection of this parasite in its mosquito intermediate host currently involves dissection and microscopic examination for larval stages. Although this method is used commonly as a screening tool for epidemiological surveys, it lacks both sensitivity and specificity. In this study, a more efficient PCR- and probe-based diagnostic assay was developed. The target selected for this assay is a segment of the 16 S rRNA gene. The assay specifically detects as little as 10 pg of D. immitis genomic DNA, equivalent to DNA derived from one third stage larva (L₃), but does not detect 100 ng (10 000-fold excess) of the purified DNA from several other filarial nematodes, including Dirofilaria striata, Dirofilaria tenuis,Dipetalonema reconditum , Wuchereria bancroftii, Brugia pahangi, B. malayi, Onchocerca volvulus or Loa loa. This assay also detects one L₃of D. immitis, the minimal biological unit of infection, in a pool of 200 mosquito heads. This assay can serve as a highly specific and sensitive tool for efficiently screening the large numbers of mosquitoes to determine, with statistical validity the seasonal transmission pattern of D. immitis in a locality prior to designing a rational preventive medication program for that parasite.     

Rapid in-situ detection kit (RisK): Development of loop-mediated isothermal amplification (LAMP) assay for the rapid identification of selected invasive alien fish in Malaysian freshwaters

Invasive alien fish species have become a silent treat towards the ecosystem especially the native fish population in Malaysia. There has been a need to develop rapid identification methods that can aid management teams in identifying fish species that are not native to our ecosystem. Current visual identification methods are highly tedious and require time, delaying action towards curbing the invasion. The LAMP assay successfully identified six popular invasive fish species in Malaysia. None of the LAMP assays showed false positives and the Limit of Detection of the LAMP primers were highly sensitive and could detect DNA samples up to 1 × 10⁻¹⁵ ng/μl. The LAMP primers designed were highly specific to the target species and did not amplify non target species. DNA sequencing was done to ensure the accuracy of LAMP assay results. This study demonstrates that LAMP is a suitable tool in species identification efforts of invasive fish species in Malaysia.     

Molecular detection and quantification of the protozoan Bonamia ostreae in the flat oyster, Ostrea edulis

Bonamia ostreae is an intracellular protozoan which is recognized as a cause of mortality in European populations of flat oysters (Ostrea edulis). Based on the recent characterization of actin genes of B. ostreae, specific primers were designed for real-time PCR using SYBR^® Green chemistry. Specificity was demonstrated by the unique melting temperature peak observed in positive samples and by the lack of amplification in samples of oysters infected by closely related parasites, including Bonamia exitiosa. A calibration curve using a cloned template was defined to estimate copy number. The assay had a 6 log- dynamic range, mean inter- and intra-assay variation coefficients of <1% and a minimum detection limit of 50 gene copies per reaction. Using infected oyster samples as templates, the assay was at least 10-fold more sensitive than conventional PCR. The quantitative assay was applied to test 132 oysters, and results were compared with the heart imprint method. There was a strong correlation between both techniques, and the results showed that the real-time PCR assay should be useful for studies of the ecology of B. ostreae and its host–parasite relationship.     

Loop mediated isothermal amplification combined with nucleic acid lateral flow strip for diagnosis of cyprinid herpes virus-3

An improved loop mediated isothermal amplification (LAMP) assay for rapid, sensitive and specific detection of cyprinid herpes virus-3 (CyHV-3), also known as koi herpes virus (KHV), was developed. The lower detection limit of the CyHV-3-LAMP assay is 10 fg DNA which equivalent to 30 copies of CyHV-3 genome. Nucleic acid lateral flow assay was used for visual detection of the LAMP products. The LAMP- nucleic acid lateral flow assay relies on DNA hybridization technology and antigen–antibody reactions in combination with LAMP. For application of this assay, the biotinylated LAMP product was hybridized with a FITC-labelled specific probe for 5 min. The resulting DNA complex could be visualised as purple band at the strip test line within 5 min of sample exposure. The nucleic acid lateral flow analysis of the LAMP product was equivalent in sensitivity but more rapid than the conventional agarose gel electrophoresis. The combination of LAMP assay with the nucleic acid lateral flow analysis can simplify the diagnosis and screening of CyHV-3 as it is simple, requires very little training, does not require specialized equipment such as a thermal cycler, the results are read visually with no need to run a gel and has a high sensitivity and specificity.     

Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) by loop-mediated isothermal amplification combined with a lateral-flow dipstick

Several methods such as traditional PCR or nested-PCR, immuno assay and histopathology have been developed for detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) formerly called monodon baculovirus (MBV). However, these methods have various disadvantages including low sensitivity, long assay time, use of toxic substances or unsuitability for field diagnosis. Loop-mediated isothermal amplification of target nucleotide sequences under isothermal conditions, combined with amplicon detection by chromatographic lateral-flow dipsticks allows for more efficient, field friendly detection within 75 min (not including DNA preparation time). In this study, the LAMP amplicon was biotinylated via an inner LAMP primer designed from a BamHI fragment B, a hypothetical protein gene of PemoNPV under isothermal condition at 63 °C for 1 h. Next, the LAMP product was hybridized at 63 °C for 5 min with an optimal FITC-labeled probe that was designed specifically for the LAMP amplicons. The FITC-labeled biotinylated LAMP product picked up gold-labeled, anti-FITC near the LFD origin and the whole, triple-labeled complex was captured by an immobilized biotin-binding protein to yield a red nano-gold stripe at the LFD test line. With a DNA template extracted from PemoNPV-infected shrimp, the LAMP–LFD detection limit was 0.1 pg, whereas one-step PCR and nested-PCR followed with gel electrophoresis was 1 pg. The LAMP–LFD method gave negative test results with buffer and DNA from shrimp infected with other common shrimp DNA viruses including, Penaeus monodon densovirus (PmDNV) formerly called hepatopancreatic parvovirus (HPV), white spot syndrome virus (WSSV) and Penaeus stylirostris densovirus (PstDNV) formerly called infectious hypodermal and hematopoietic necrosis virus (IHHNV). The test platform can be adapted easily for rapid detection of other shrimp viruses, since the LAMP–LFD combination system was a highly sensitive, specific, convenient, and does not require sophisticated instruments.     

Development of a pair of real-time loop mediated isothermal amplification assays for detection of Yersinia pestis,the causative agent of plague

Yersinia pestis, the causative agent of plague mainly infects rodents, while humans are the accidental host. The conventional diagnostic methods available for Y. pestis exhibit cross-reactivity with other enteropathogenic bacteria which makes its detection difficult. Rapid and reliable point-of-care detection of Y. pestis is essential for timely initiation of medical treatment. In the present study, a pair of loop mediated isothermal amplification (LAMP) assays has been developed for rapid detection of Y. pestis. Two sets of LAMP primers, each containing 6 primers were specifically designed targeting caf1 and 3a genes located on pFra plasmid and chromosome of Y. pestis, respectively. Isothermal amplification was accomplished at 65 °C for 40 min for caf1 target, and at 63 °C for 50 min for 3a choromosomal target. The analytical sensitivity of the assay for the caf1 and 3a targets was found to be 500 fg and 100 fg genomic DNA of Y. pestis, respectively. The caf1 and 3a LAMP assays detected as few as 100 copies of caf1 and 10 copies of 3a gene targets harboured in the respective recombinant plasmids. The amplified products were detected visually under visible and UV light using SYBR Green 1 dye. The assay pair was found to be highly specific as it did not cross-react with closely related and other bacterial species.     

Development and clinical validation of a loop-mediated isothermal amplification method for the rapid detection of Neisseria meningitidis

Loop-mediated isothermal amplification (LAMP) is an innovative technique that allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, optimization, and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detection of capsular Neisseria meningitidis were described. Highly specific detection of capsular N. meningitidis type strains and clinical isolates was demonstrated, with no cross-reactivity with other Neisseria spp. or with a comprehensive panel of other common human pathogens. The lower limit of detection was 6 ctrA gene copies detectable in 48 min, with positive reactions readily identifiable visually via a simple color change. Higher copy numbers could be detected in as little as 16 min. When applied to a total of 394 clinical specimens, the LAMP assay in comparison to a conventional TaqMan® based real-time polymerase chain reaction system demonstrated a sensitivity of 100% and a specificity of 98.9% with a κ coefficient of 0.942. The LAMP method represents a rapid, sensitive, and highly specific technique for the detection of N. meningitidis and has the potential to be used as a point-of-care molecular test and in resource-poor settings.     

Specific detection and quantification of the phytopathogenic agent ‘Candidatus Phytoplasma prunorum’

‘Candidatus Phytoplasma prunorum’ is a wall-less bacterium associated with European stone fruit yellows (ESFY), a severe disease of Prunus spp. (mainly apricot and Japanese plum trees). It can be spread by one insect vector, Cacopsylla pruni, and by the trade of infected material. The availability of PCR-based methods allowing a sensitive and specific detection of ‘Ca. P. prunorum’ is crucial for this phytoplasma because, at present, it is uncultured and cannot be detected serologically. We developed a PCR test which, in contrast to the existing detection tools, provides a fast, specific and sensitive detection of ‘Ca. P. prunorum’ in plants and insects. For studies requiring an absolute quantification of the phytoplasma titer, the same primers were used to develop a real-time PCR assay, including a standard for C. pruni. The sensitivity of these molecular tools was compared by serial dilutions and their specificity was assessed both in silico and experimentally for reference strains and field samples of the closely related phytoplasma ‘Ca. P. prunorum’, ‘Ca. P. pyri’ (pear decline agent) and ‘Ca. P. mali’ (apple proliferation agent), as well as for representative strains of the ‘Ca. Phytoplasma’ genus.     

Evaluation of loop-mediated isothermal amplification for detection of Toxoplasma gondii in water samples and comparative findings by polymerase chain reaction and immunofluorescence test (IFT)

The development and evaluation of a 1-step single-tube accelerated loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Toxoplasma in water samples is described. The method has been evaluated based on the amplification of B1 and TgOWP Toxoplasma genes, and it demonstrated a sensitivity detection limit of 0.1 tachyzoites' DNA for both genes. LAMP detection was evaluated and compared with nested polymerase chain reaction (PCR) in 26 water sample pellets spiked with known numbers of Toxoplasma oocysts. After DNA extraction, the detection sensitivity in spiked pellets was 100% by LAMP and 53.8% by PCR. Subsequently, 52 natural water samples of different origin were directly investigated by 3 assays: LAMP, PCR, and immunofluorescence test (IFT). Twenty-five (48%) of 52 have been found positive for Toxoplasma DNA by LAMP, whereas nested PCR products were generated in 7 of 52 (13.5%) water samples. All 52 water samples were negative for Toxoplasma by IFT. These data clearly indicate LAMP as a rapid, specific, and sensitive tool for the detection of Toxoplasma contamination in water samples.     

Visual detection of white spot syndrome virus using DNA-functionalized gold nanoparticles as probes combined with loop-mediated isothermal amplification

The integration of loop-mediated isothermal amplification (LAMP) and DNA-functionalized AuNPs as visual detection probes (LAMP–AuNPs) was developed and applied for the detection of white spot syndrome virus (WSSV) from Penaeid shrimp in this study. The principle of this combination assay relies on the basis of stability characteristics of the DNA-functionalized AuNPs upon hybridization with the complementary target DNA toward salt-induced aggregation. If the detected target DNA is not complementary to the ssDNA probes, the DNA-functionalized AuNPs will be aggregated due to the screening effect of salt, resulting in the change of solution color from red to blue/gray and shift of the surface plasmon peak to longer wavelength. While the DNA-functionalized AuNPs are perfectly matched to the detected target DNA, the color of solution still remains red in color and no surface plasmon spectral shift. This assay provides simply technique, time-saving and its detection results could be achieved qualitatively and quantitatively by visualization using the naked eye due to the colorimetric change and by measurement using the UV–vis spectroscopy due to the surface plasmon spectral shift, respectively. In this study, LAMP–AuNPs assay was successfully developed with the detection of WSSV-LAMP generated product at 0.03 μg/reaction, and showed the sensitivity of 2 × 10² copies WSSV plasmid DNA, that is comparable to the most sensitive method reported to date. The LAMP–AuNPs assay described in this study revealed a highly sensitive, rapid and reliable diagnostic protocol for detection of WSSV. This technique has a potential as a routine method for assessing the infectious diseases in Penaeid shrimp not only for WSSV, but also for other shrimp pathogens, and can be useful tool in field conditions for the diagnosis or surveillance programs.     

Hemoculture and Direct Sputum Detection of mecA‐Mediated Methicillin‐Resistant Staphylococcus aureus by Loop‐Mediated Isothermal Amplification in Combination With a Lateral‐Flow Dipstick

This study reports loop‐mediated isothermal amplification (LAMP) for rapid detection of methicillin‐resistant Staphylococcus aureus from direct clinical specimens. Four primers including outer and inner primers were specifically designed on the two target sequences—femB to identify S. aureus and mecA to identify antibiotic‐resistant gene. Reference strains including various species of gram‐positive/gram‐negative isolates were used to evaluate and optimize LAMP assays. The optimum LAMP condition was found at 63°C within 70 min assay time (include hybridization with FITC probe for 5 min and further 5 min for reading the results on the lateral flow dipstick). The detection limits of LAMP for mecA was 10 pg of total DNA or 100 CFU/ml. The LAMP assays were applied to a total of 155 samples of direct DNA extraction from sputum and hemoculture bottles. The sensitivity of LAMP for mecA detection in sputum and hemoculture bottles was 93.3% (28/30) and 100% (52/52), respectively. In conclusion, LAMP assay is an alternative technique for rapid detection of MRSA infection with a technical simplicity and cost‐effective method in a routine diagnostic laboratory.