Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus

Background

Microbial cell-cell interactions in the oral flora are believed to play an integral role in the development of dental plaque and ultimately, its pathogenicity. The effects of other species of oral bacteria on biofilm formation and virulence gene expression by Streptococcus mutans, the primary etiologic agent of dental caries, were evaluated using a dual-species biofilm model and RealTime-PCR analysis.

Results

As compared to mono-species biofilms, biofilm formation by S. mutans was significantly decreased when grown with Streptococcus sanguinis, but was modestly increased when co-cultivated with Lactobacillus casei. Co-cultivation with S. mutans significantly enhanced biofilm formation by Streptococcus oralis and L. casei, as compared to the respective mono-species biofilms. RealTime-PCR analysis showed that expression of spaP (for multi-functional adhesin SpaP, a surface-associated protein that S. mutans uses to bind to the tooth surface in the absence of sucrose), gtfB (for glucosyltransferase B that synthesizes α1,6-linked glucan polymers from sucrose and starch carbohydrates) and gbpB (for surface-associated protein GbpB, which binds to the glucan polymers) was decreased significantly when S. mutans were co-cultivated with L. casei. Similar results were also found with expression of spaP and gbpB, but not gtfB, when S. mutans was grown in biofilms with S. oralis. Compared to mono-species biofilms, the expression of luxS in S. mutans co-cultivated with S. oralis or L. casei was also significantly decreased. No significant differences were observed in expression of the selected genes when S. mutans was co-cultivated with S. sanguinis.

Conclusions

These results suggest that the presence of specific oral bacteria differentially affects biofilm formation and virulence gene expression by S. mutans.     

Rapid identification of Plasmodium-carrying mosquitoes using loop-mediated isothermal amplification

With an aim to develop a quick and simple method to survey pathogen-transmitting vectors, LAMP (loop-mediated isothermal amplification) was applied to the identification of Plasmodium-carrying mosquitoes, specifically a Plasmodium-transmitting experimental model using rodent malaria parasite (Plasmodium berghei) and anopheline mosquitoes (Anopheles stephensi). The detection sensitivity limit of the LAMP reaction amplifying the SPECT2 gene was determined to be 1 × 10² purified Plasmodium parasites, estimated to be sufficient for reliable identification of infectious mosquitoes. The robustness of the LAMP reaction was revealed by its ability to detect both Plasmodium oocysts and sporozoites from an “all-in-one” template using whole mosquito bodies. Moreover, LAMP successfully identified an infectious mosquito carrying just a single oocyst in its midgut, a level that can be easily overlooked in conventional microscopic analysis. These observations suggest that LAMP is more reliable and useful for routine diagnosis of vector mosquitoes in regions where vector-borne diseases such as malaria are endemic.     

Real-time quantitative loop-mediated isothermal amplification as a simple method for detecting white spot syndrome virus

Aims:  White spot syndrome virus (WSSV) continues to be the most pathogenic virus among the crustacean aquaculture causing mass mortality. In the present study, we established a one-step, single tube, real-time accelerated loop-mediated isothermal amplification (real-time LAMP) for quantitative detection of WSSV.
Materials and Methods:  A set of six specially designed primers that recognize eight distinct sequences of the target. The whole process can be completed in 1 h under isothermal conditions at 63°C. Detection and quantification can be achieved by real-time monitoring in an inexpensive turbidimeter based on threshold time required for turbidity in the LAMP reaction. A standard curve was constructed by plotting viral titre against the threshold time ( T _t) using plasmid standards with high correlation coefficient ( R ² = 0·988).
Conclusions:  Sensitivity analysis using 10-fold dilutions (equivalent to 35 ng  μ l⁻¹ to 35 ag  μ l⁻¹) of plasmid standards revealed this method is capable of detecting upto 100 copies of template DNA. Cross-reactivity analysis with DNA/cDNA of IHHNV, TSV, YHV-infected and healthy shrimp showed this method is highly specific for quantitative detection of WSSV.
Significance and Impact of the Study:  WSSV real-time LAMP assay appears to be precise, accurate and a valuable tool for the detection and quantification of WSSV in large field samples and epidemiological studies.     

环介导等温扩增快速检测B群链球菌的临床应用

目的建立采用环介导等温扩增（LAMP）技术从临床标本中快速检测B群链球菌的方法。方法根据美国国家生物信息中心上提交的B群链球菌的cfb基因序列（登陆号X72754）设计特异LAMP引物,以热裂解法提取标本中细菌的DNA,然后以LAMP技术扩增cfb基因来鉴定其是否为B群链球菌。在采用LAMP技术检测B群链球菌的同时,以选择性培养法和PCR技术平行检测相同标本,并将3种方法的检测结果比较。结果在176例阴道拭子和176例直肠拭子中,选择性培养法检测到的B群链球菌例数为49和61、LAMP法检测到的B群链球菌例数为49和59,PCR法检测到的B群链球菌例数为48和58。以选择性培养法为金标准,LAMP法检测B群链球菌的灵敏度和特异度分别为96．7％和100％,PCR法检测B群链球菌的灵敏度和特异度分别为95．1％和100％。LAMP法检测B群链球菌的时间为2h左右,模拟菌株的检测结果显示该方法的最低检测限为10^2CFU／mL。结论该方法灵敏度高,特异性强,操作方便简单,适合从临床标本中快速检测B群链球菌。     

Isolation of single-stranded DNA from loop-mediated isothermal amplification products.

Loop-mediated isothermal amplification (LAMP), in which a specific DNA sequence can be directly amplified under isothermal conditions, yields DNA in large quantities of more than 500 microg/ml. We have developed a method to isolate single-stranded DNA fragments from LAMP products that are stem-loop DNAs with several inverted repeats of the target DNA. This method requires the TspRI restriction enzyme, a primer hybridized to the 3' overhanging sequence at its cleavage site, and a DNA polymerase with strand displacement activity. The LAMP products are digested with TspRI and are then extended using the primer, producing the strand-specific DNA fragments. All processes, from LAMP reaction to primer extension, can be carried out at the same temperature. The use of strand-specific DNA would be conducive for detection by hybridization technique such as DNA microarrays.     

Transmission dynamics of lymphatic filariasis: density-dependence in the uptake of Wuchereria bancrofti microfilariae by vector mosquitoes

Gaining a better understanding of parasite infection dynamics in the vector mosquito (Diptera: Culicidae) population is central to improving knowledge regarding the transmission, persistence and hence control of lymphatic filariasis. Here, we use data on mosquito feeding experiments collated from the published literature to examine the available evidence regarding the functional form of the first component of this parasite-vector relationship for Wuchereria bancrofti (Filarioidea: Onchocercidae) causing Bancroftian filariasis, i.e. the rate of microfilariae (mf) uptake from the blood of infected humans by the feeding mosquito vector. Using a simple logarithmic regression model for describing the observed relationships between the mean numbers of mf ingested per mosquito and parasite load in humans in each study, and a linear mixed-effects meta-analytical framework for synthesizing the observed regressions across studies, we show here for the first time clear evidence for the existence of density-dependence in this process for all the three major filariasis transmitting mosquito vectors. An important finding of this study is that this regulation of mf uptake also varies significantly between the vector genera, being weakest in Culex, comparatively stronger in Aedes and most severe and occurring at significantly lower human mf loads in Anopheles mosquitoes. The analysis of the corresponding mf uptake prevalence data has further highlighted how density-dependence in mf uptake may influence the observed distributions of mf in vector populations. These results show that whereas strong regulation of mf uptake, especially when it leads to saturation in uptake at low human parasite intensities, can lead to static distributions of mf per mosquito with host parasite intensity, a weaker regulation of mf ingestion can give rise to changes in both mean mf loads and in the frequency distribution of parasites/mosquito with increasing human parasite intensity. These findings highlight the importance of considering local vector infection dynamics when attempting to predict the impacts of community-based filariasis control. They also emphasize the value of developing and applying robust meta-analytic methods for estimating functional relationships regarding parasitic infection from population ecological data.     

Development of a loop-mediated isothermal amplification (LAMP) method for specific detection of Mycobacterium bovis

Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection of M. bovis is fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective against M. bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification of M. bovis by targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely in M. bovis. The assay''s specificity was evaluated using 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven M. tuberculosis complex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected only M. bovis isolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies of M. bovis genomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection of M. bovis infections in cattle and humans in resource-limited areas.     

Development and evaluation of loop-mediated isothermal amplification for rapid detection of Haemophilus parasuis

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions and has a high specificity and efficiency. We developed a LAMP assay targeting the 16S rRNA gene for rapid detection of Haemophilus parasuis. The results obtained from testing 31 H. parasuis strains and 28 other bacterial species strains showed that LAMP was as specific as, and more sensitive than, nested PCR. Fifty-five lung samples were collected from 55 healthy pigs. All the samples were negative for H. parasuis by bacterial isolation, nested PCR and LAMP, respectively. In addition, 122 lung samples were collected from 122 pigs with apparent respiratory problems. Sixty-five were positive by bacterial isolation. All the samples that were positive by bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated higher sensitivity than nested PCR, picking up 16 additional cases. The LAMP assay also gave a same result compared with the nested PCR when the two assays were used, respectively, to detect H. parasuis from samples obtained from experimentally infected pigs. We concluded that LAMP is a highly sensitive and reliable method for detection of H. parasuis infection.     

Development of a loop-mediated isothermal amplification assay for porcine circovirus type 2

In this study,the loop-mediated isothermal amplification(LAMP)method was used to develop a rapid and simple detection system for porcine circovirus type 2(PCV2).According to the PCV2 sequences published in GenBank,multiple LAMP primers were designed targeting conserved sequences of PCV2.Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template,LAMP reactions in a PCV2 LAMP system was performed,the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye.The results showed highly-efficient and specific amplification in 30 min at 63℃ with a LAMP real-time turbidimeter.Furthermore,PCV2 DNA templates,with a detection limit of 5.5×10-5ng of nucleic acid,indicated that this assay was highly sensitive.The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter,showing the potential simplicity of interpretation of the assay results.The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples.In addition it offers higher specificity and sensitivity,shorter reaction times and simpler procedures than the currently available methods of PCV2 detection.It is therefore a promising tool for the effective and efficient detection of PCV2.     

Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for periodontal pathogen, Porphyromonas gingivalis. A set of six primers was designed by targeting the 16S ribosomal RNA gene. By the detection system, target DNA was amplified and visualized on agarose gel within 30 min under isothermal condition at 64 degrees C with a detection limit of 20 cells of P. gingivalis. Without gel electrophoresis, the LAMP amplicon was directly visualized in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. Detection limits of these naked-eye inspections were 20 cells and 200 cells, respectively. Although false-positive DNA amplification was observed from more than 10(7) cells of Porphyromonas endodontalis, no amplification was observed in other five related oral pathogens. Further, quantitative detection of P. gingivalis was accomplished by a real-time monitoring of the LAMP reaction using SYBR Green I with linearity over a range of 10(2)-10(6) cells. The real-time LAMP was then applied to clinical samples of dental plaque and demonstrated almost identical results to the conventional real-time PCR with an advantage of rapidity. These findings indicate the potential usefulness of LAMP for detecting and quantifying P. gingivalis, especially in its rapidity and simplicity.     

Development and application of a loop-mediated isothermal amplification method on rapid detection of Pseudomonas aeruginosa strains

We developed and evaluated the specificity and sensitivity of a simple loop-mediated isothermal amplification (LAMP) method for rapid detection of P. aeruginosa strains. The optimal reaction condition was found to be 65°C for 45 min, with the detection limit as 100 fg DNA/tube and 10 CFU/reaction. Application of LAMP assays were performed 426 clinical samples (including 252 P. aeruginosa and 174 non- P. aeruginosa isolates) using a rapid procedure and easy result confirmation. Sensitivity of LAMP and PCR assays was found to be 97.6% (246/252) and 90.5% (228/252), respectively; with a 100% specificity for both assays.     

Detection of phytoplasma by loop-mediated isothermal amplification of DNA (LAMP)

Napier stunt phytoplasma (16SrXI and 16SrIII) in eastern Africa is a serious threat to the expansion of Napier grass (Pennisetum purpureum) farming in the region, where it is widely cultivated as fodder in zero grazing livestock systems. The grass has high potential for bio-fuel production, and has been adopted by farmers as a countermeasure to cereal stem borer Lepidoptera, since it attracts and traps the insect. Diagnosis of stunt phytoplasma have been largely by nested polymerase chain reaction (nPCR) targeting the 16S rRNA gene. However, the method is laborious, costly and technically demanding. This investigation has developed a simpler but effective phytoplasma diagnostic tool, called; loop-mediated isothermal amplification of DNA (LAMP). The assay was tested on 8 symptomatic and 8 asymptomatic plants, while its detection limit was compared to nested PCR using samples serially diluted from 3 ng/μl to 0.38 pg/μl. Molecular typing of LAMP products was determined by BsrI restriction digestion and Southern blot analysis. The assay sensitivity, positive and negative predictive values were estimated, while the specificity was tested on 11 phytoplasma groups. LAMP was specific to 5 phytoplasma groups: 16SrVI, X, XI and XVI. BsrI restriction digestion produced two predicted fragments, and there was specific binding of probe DNA to the LAMP amplicons in Southern blot analysis. The assay sensitivity was 100%, while the positive and negative predictive values were 63 and 100% respectively. LAMP was 20-fold more sensitive than nested PCR. This study validates LAMP for routine diagnosis of Napier stunt and other closely related phytoplasmas.     

Real-time quantitative LAMP (loop-mediated isothermal amplification of DNA) as a simple method for monitoring ammonia-oxidizing bacteria

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique for the amplification of DNA under isothermal conditions. For the first time, we applied this method to develop a simple and quantitative monitoring method for environmental microorganisms targeting amoA gene in ammonia-oxidizing bacteria. Quantitative analysis was performed first by measuring fluorescence derived from an intercalation dye using a real-time thermal cycler, and then by measuring the turbidity of the reaction solution using a real-time turbidimeter. As a result, it was possible to quantify the initial amoA DNA concentration from an environment with a sensitivity down to 10(2) DNA copies of target DNA and a dynamic range of 7-9 orders in magnitude. Background DNA from nontargeted bacteria (Pseudomonas denitrificans) that does not encode amoA gene did not affect the quantitative capability of LAMP. Over results suggested that the real-time LAMP is effective for monitoring microorganisms and their gene expression in environments.     

唐菖蒲伯克霍尔德氏菌椰毒假单胞菌酵米面亚种环介导等温扩增荧光方法的建立

【目的】建立一个基于环介导等温扩增与荧光探针结合的唐菖蒲伯克霍尔德氏菌椰毒假单胞菌酵米面亚种(Burkholderia gladioli pv. cocovenenans)特异性检测方法。【方法】通过多重序列比对分析,选择B. gladioli pv. cocovenenans共有的bonA为靶位点,设计环介导等温扩增的特异性引物和荧光探针,通过19株B. gladioli和3株非目标菌株,验证该方法的特异性。将该方法的检测特异性与现有的基于实时荧光定量PCR鉴定B. gladioli pv. cocovenenans的方法进行比较,并对建立的方法进行灵敏度探究。【结果】建立的环介导等温扩增荧光鉴定方法对B. gladioli pv.cocovenenans具有特异性,能在30 min内得出结果,且该方法能进行可视化直接观测。该方法的基因组DNA最低检测限度为1.98pg/μL,检测灵敏度为2.7×10²CFU/mL。另外,该方法能应用到食品样品中B. gladioli pv. cocovenenans的检测。【结论】本研究建立了一个快速、可视化、特异性强的检测方...     

Rapid, sensitive and simple detection method for koi herpesvirus using loop-mediated isothermal amplification

New methods were developed for the detection of koi herpesvirus (KHV, CyHV-3) by LAMP, which were compared with the PCR for specificity and sensitivity. We designed two primer sets targeting a specific sequence within the 9/5 PCR amplicon (9/5 LAMP) and the upper region of the Sph I-5 PCR amplicon ( Sph I-5 LAMP), including a sequence highly conserved among the strains. The amplification was monitored in real-time based on the increase in turbidity, with magnesium pyrophosphate as the by-product. The reactions were carried out under isothermal conditions at 65°C for 60 min. The detection limit of both LAMP was six copies, equal to the modified Sph I-5 PCR. No cross-reactivity with other fish pathogenic viruses and bacteria was observed. Sph I-5 LAMP was found to have a quicker response in terms of the reaction velocity than 9/5 LAMP. Therefore, we consider Sph I-5 LAMP to be superior for routine use. Additionally, LAMP was found applicable to crude extract from gills and other organs. LAMP methods are superior in terms of sensitivity, specificity, rapidity and simplicity, and are potentially a valuable diagnostic tool for KHV infections.     

Assessing density dependence in the transmission of lymphatic filariasis: uptake and development of Wuchereria bancrofti microfilariae in the vector mosquitoes

Understanding density dependence in the transmission of lymphatic filariasis is essential for assessing the prospects of elimination. This study seeks to quantify the relationship between microfilaria (Mf) density in human blood and the number of third stage (L3) larvae developing in the mosquito vectors Aedes polynesiensis Marks and Culex quinquefasciatus Say (Diptera: Culicidae) after blood-feeding. Two types of curves are fitted to previously published data. Fitting a linearized power curve through the data allows for correction for measurement error in human Mf counts. Ignoring measurement error leads to overestimation of the strength of density dependence; the degree of overestimation depends on the accuracy of measurement of Mf density. For use in mathematical models of transmission of lymphatic filariasis, a hyperbolic saturating function is preferable. This curve explicitly estimates the Mf uptake and development at lowest Mf densities and the average maximum number of L3 that can develop in mosquitoes. This maximum was estimated at 23 and 4 for Ae. polynesiensis and Cx. quinquefasciatus, respectively.