TC-RT-PCR检测菜豆荚斑驳病毒的研究

根据已报道的菜豆荚斑驳病毒(Bean pod mottle virus,BPMV)外壳蛋白(Coat protein,CP)基因序列设计特异性引物,应用试管捕捉RT-PCR(Tube capture RT-PCR,TC-RT-PCR)技术对大豆种皮上的BPMV进行检测.结果表明,TC-RT-PCR方法能从携带BPMV的大豆种皮上扩增到预期大小的基因片段.将TC-RT-PGR扩增产物克隆测序后进行序列分析,结果显示扩增到的片段序列与BPMV的CP基因序列具有高度同源性,进一步证实了该方法的准确性.应用TC-RT-PCR方法,成功检测了一批进境大豆样品.本文建立的TC-RT-PCR方法,为大豆种子上BPMV的检测和诊断提供了一种新的参考方法.     

Rapid detection of Brucella spp. by the loop-mediated isothermal amplification method

Aims:  To develop a rapid and sensitive method for detecting Brucella spp.
Methods and Results:  Two sets of six Brucella -specific primers for loop-mediated isothermal amplification (LAMP) were designed from the sequence of the Brucella abortus BCSP31 gene. The specificity and sensitivity were examined for six Brucella species (22 strains) and 18 non- Brucella species (28 strains). The LAMP assay was specific to Brucella spp. in 35 min at 63°C and sensitive (detected 10 fg of genomic DNA). The assay was also applied for the detection of Brucella DNA in contaminated milk and infected mouse organs.
Conclusions:  We developed a sensitive and specific LAMP assay for Brucella spp., with the test appearing to be useful for the detection of the pathogen from clinical and food samples.
Significance and Impact of the Study:  This is the first report of the development of LAMP for the detection of Brucella spp. As the LAMP assay can be performed at a constant temperature and its reactivity is directly observed with the naked eye without electrophoresis, our assay should be useful for the diagnosis of brucellosis as well as the detection of the bacteria in environmental or food samples.     

应用环介导等温扩增技术(LAMP)快速检测家蚕核型多角体病毒的研究

由家蚕核型多角体病毒(Bombyx mori nucleopoyhedrosis virus,BmNPV)侵染家蚕Bombyx mori引起的家蚕核型多角体病(血液型脓病)在养蚕生产中发生较为普遍,对蚕业生产造成重大的经济损失。本研究建立了快捷有效的环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)用于快速检测BmNPV,为蚕业生产提供了一个有效检测BmNPV和进行早期诊断的技术。该方法是针对BmNPV的pe38基因的6个区段设计的6条引物用于扩增检测,整个反应在恒温条件63℃下进行25 min,扩增产物用电泳法和可视法检测(用SYBR Green I染色)。结果显示,LAMP检测方法的灵敏度是常规PCR方法的100倍,能检测的范围为21个拷贝。另外,用4.86×108OBs/mL感染4龄起蚕,提取血淋巴DNA为模板,PCR在感染36 h后检出病毒DNA,LAMP法能检测感染12h后的样品。     

Rapid detection of Trichinella spiralis larvae in muscles by loop-mediated isothermal amplification

Trichinella spiralis is a tissue-dwelling nematode parasite. A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the sensitive and rapid detection of T. spiralis larvae in muscle samples. Sixteen sets of primers were designed to recognise distinct sequences of a conserved gene, a 1.6 kb repetitive element of the Trichinella genome. One set of primers was selected as the most appropriate for rapid detection. The specificity and sensitivity of the primers in LAMP reactions for T. spiralis larvae and muscle samples of mice infected with T. spiralis were determined. Another 10 heterologous parasites were selected for specificity assays. The results showed that target DNA was amplified and visualised by monitoring turbidity and adding calcein detection methods within 70 min at an isothermal temperature of 63 °C. The sensitivity of LAMP with the detection limit of 362 fg/μl was >10 times higher than that for PCR. The designed primers had a good specificity. No cross-reactivity was found with the DNA of any other parasites. The assay was able to detect T. spiralis in all mouse muscle samples infected with 10 T. spiralis larvae on day 20 p.i. We believe this is the first report regarding the application of the LAMP assay for detection of T. spiralis larvae in muscle samples from experimentally infected mice. This method demonstrates a potentially valuable means for the direct detection of T. spiralis larvae in meat inspection.     

Temporal and spatial Bean pod mottle virus‐induced gene silencing in soybean

Virus‐induced gene silencing (VIGS) is a powerful reverse genetics tool in plant science. In this study, we investigated the temporal and spatial silencing patterns achieved by Bean pod mottle virus (BPMV)‐based VIGS in soybean using virus constructs targeting green fluorescence protein (GFP). Silencing GFP enabled an in‐depth analysis of silencing in soybean tissues over time in a transgenic line constitutively expressing GFP. We discovered evidence for variable GFP silencing based on insert orientation and targeted region in the coding sequence. A 3′ sequence in reverse orientation produced the strongest silencing phenotypes. Furthermore, we documented that BPMV VIGS can achieve widespread silencing in a broad range of tissues, including leaves, stems, flowers and roots. Near‐complete silencing was attained in leaves and flowers. Although weaker than in shoots, the observed gene silencing in soybean roots will also allow reverse genetics studies in this tissue. When GFP fluorescence was assayed in cross‐sections of stems and leaf petioles, near‐complete and uniform silencing was observed in all cell types. Silencing was observed from as early as 2 weeks post‐virus inoculation in leaves to 7 weeks post‐virus inoculation in flowers, suggesting that this system can induce and maintain silencing for significant durations.     

Rapid and sensitive detection of Singapore grouper iridovirus by loop-mediated isothermal amplification

Aims:  The aim of this paper was to develop a loop-mediated isothermal amplification (LAMP) method for rapid, sensitive and inexpensive detection of Singapore grouper iridovirus (SGIV) in grouper (GP), Epinephelus sp.
Methods and Results:  A set of six specific primers was designed by targeting the SGIV ORF-014L. With Bst DNA polymerase large fragment, the target DNA can be amplified as early as 20 min at 65°C in a simple water bath. The detection limit is about 0·02 fg (equivalent to 6·3 copies) of plasmid ORF-014L. LAMP products could be judged with three different methods. There were no cross-reactions with seven other aquatic animal viruses indicating high specificity of the LAMP. The LAMP method was applied to detect SGIV in virus-infected GP cells and GP tissues effectively.
Conclusions:  The LAMP described in this study is a cheap, sensitive, specific and rapid protocol for the detection of SGIV in cells and in GP tissues.
Significance and Impact of the Study:  The developed LAMP method can be simply applied both in field condition and in laboratory operation for specific detection of SGIV infection.     

Detection of Botrytis cinerea by loop-mediated isothermal amplification

Aims: To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop‐mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting. Methods and Results: A LAMP assay was designed based on the intergenic spacer of the B. cinerea nuclear ribosomal DNA (rDNA). The resulting assay was characterized in terms of sensitivity and specificity using DNA extracted from cultures. The assay consistently amplified 65 pg B. cinerea DNA. No cross‐reactivity was observed with a range of other fungal pathogens, with the exception of the closely related species Botrytis pelargonii. Use of a novel real‐time LAMP platform (the OptiGene Genie I) allowed detection of B. cinerea in infected rose petals, with amplification occurring in <15 min. Conclusions: The LAMP assay that was developed is suitable for rapid detection of B. cinerea in infected plant material. Significance and Impact of the Study: The LAMP method combines the sensitivity and specificity of nucleic acid‐based methods with simplified equipment and a reduced reaction time. These features make the method potentially suitable for on‐site use, where the results of testing could help to inform decisions regarding the storage and processing of commodities affected by B. cinerea, such as cut flowers, fruit and vegetables.     

Detection of fish nocardiosis by loop-mediated isothermal amplification

AIMS: Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a protocol for detecting Nocardia seriolae which is a causative agent of fish nocardiosis, was designed. METHODS AND RESULTS: A set of four primers, two inner and two outer, were designed based on the sequence of the 16S-23S ribosomal RNA internal transcribed spacer region of N. seriolae. Time and temperature conditions for detection of N. seriolae were optimized for 60 min at 65 degrees C. Other fish pathogen was not amplified by this LAMP system. The detection of N. seriola using LAMP was found to be more sensitive than that by polymerase chain reaction. CONCLUSIONS: LAMP is a highly sensitive and rapid diagnostic procedure for detection of N. seriolae. SIGNIFICANCE AND IMPACT OF THE STUDY: LAMP is a useful diagnostic method for fish nocardiosis.     

Silencing genes encoding omega-3 fatty acid desaturase alters seed size and accumulation of Bean pod mottle virus in soybean

Omega-3 fatty acid desaturase (FAD3)-catalyzed conversion of linoleic acid to linolenic acid (18:3) is an important step for the biosynthesis of fatty acids as well as the phytohormone jasmonic acid (JA) in plants. We report that silencing three microsomal isoforms of GmFAD3 enhanced the accumulation of Bean pod mottle virus (BPMV) in soybean. The GmFAD3-silenced plants also accumulated higher levels of JA, even though they contained slightly reduced levels of 18:3. Consequently, the GmFAD3-silenced plants expressed JA-responsive pathogenesis-related genes constitutively and exhibited enhanced susceptibility to virulent Pseudomonas syringae. Increased accumulation of BPMV in GmFAD3-silenced plants was likely associated with their JA levels, because exogenous JA application also increased BPMV accumulation. The JA-derived increase in BPMV levels was likely not due to repression of salicylic acid (SA)-derived signaling because the GmFAD3-silenced plants were enhanced in SA-dependent defenses. Furthermore, neither exogenous SA application nor silencing the SA-synthesizing phenylalanine ammonia lyase gene altered BPMV levels in soybean. In addition to the altered defense responses, the GmFAD3-silenced plants also produced significantly larger and heavier seed. Our results indicate that loss of GmFAD3 enhances JA accumulation and, thereby, susceptibility to BPMV in soybean.     

应用环介导等温扩增技术(LAMP)检测蜜蜂黑蜂王台病毒的研究

【目的】本文旨在建立用于临床检测黑蜂王台病毒(Black queen cell virus,BQCV)的环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP),为该疾病的检测和防控提供技术支撑。【方法】根据BQCV基因保守序列设计4条特异性引物,探究LAMP扩增的最优条件,并与常规的PCR(Polymerase chain reaction)检测方法进行比较。【结果】建立的LAMP方法特异性好,检测下限为86 fg,灵敏度比PCR高100倍。临床检测显示其对意大利蜜蜂Apis mellifera ligustica和中华蜜蜂Apis cerana cerana均准确有效,且检出率比普通PCR法高10%~20%。【结论】建立的针对BQCV的LAMP检测方法为养蜂生产第一线检测和预防BQCV提供了技术支持,有一定的应用价值。     

Rapid detection of Shigella and Salmonella in rhesus monkeys by loop-mediated isothermal amplification assay

Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting bacterial diseases in nonhuman primate laboratory animals. Materials and Methods A total of 205 fecal samples of rhesus monkeys were detected in this LAMP assay. The specificity and sensitivity of LAMP for Shigella and Salmonella were analyzed,and real-time polymerase chain reaction( REAL-TIME PCR) assay was employed as control. Results The LAMP method established here needed only 45 min to complete the reaction at 63℃. Its detection limit was 10 pg / μL and with a high specificity. The positive rate of Shigella and Salmonella was 1. 5% and 6. 3%,respectively. Conclusions Here we have established a fast and simple Shigella and Salmonella LAMP detection method that has strong specificity and high sensitivity and is suitable for rapid detection of bacterial disease in macaques. The development of this rapid detection kit is underway,and it will be helpful to the diarrhea detection.     

Sensitive and rapid detection of edwardsiellosis in fish by a loop-mediated isothermal amplification method

Here we report a rapid and sensitive method (using loop-mediated isothermal amplification [LAMP]) for the diagnosis of edwardsiellosis, a fish disease caused by Edwardsiella tarda, in Japanese flounder. A set of four primers was designed, and conditions for the detection were optimized for the detection of E. tarda in 45 min at 65 degrees C. No amplification of the target hemolysin gene was detected in other related bacteria. When the LAMP primers were used, detection of edwardsiellosis in infected Japanese flounder kidney, and spleen and seawater cultures was possible. We have developed a rapid and sensitive diagnostic protocol for edwardsiellosis detection in fish. This is the first report of the application of LAMP for the diagnosis of a fish pathogen.     

Simple and specific detection of Bordetella holmesii by using a loop-mediated isothermal amplification assay

A loop-mediated isothermal amplification (LAMP) assay for simple detection of Bordetella holmesii was developed. This assay discriminates between B. holmesii and other Bordetella species and successfully detect B. holmesii DNA in nasopharyngeal swab samples from subjects with suspected pertussis. The LAMP assay results were in complete agreement with the results of previously published real-time PCR assay, indicating that the former is a powerful tool for the accurate diagnosis and surveillance of B. holmesii.     

Rapid detection of viable salmonellae in produce by coupling propidium monoazide with loop-mediated isothermal amplification

Recent outbreaks linked to Salmonella-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMA-LAMP). We first designed and optimized a LAMP assay targeting Salmonella. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killed Salmonella cells with concentrations up to 10(8) CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viable Salmonella cells in pure culture and 6.1 × 10(3) to 6.1 × 10(4) CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (T(T)) values and viable Salmonella cell numbers was high (R(2) = 0.949 to 0.993), with a quantification range (10(2) to 10(5) CFU/reaction in pure culture and 10(4) to 10(7) CFU/g in produce) comparable to that of PMA in combination with quantitative real-time PCR (PMA-qPCR). The complete PMA-LAMP assay took about 3 h to complete when testing produce samples. In conclusion, this rapid, accurate, and simple method to detect and quantify viable Salmonella cells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce.     

Rapid detection of Pseudomonas aeruginosa in mouse feces by colorimetric loop-mediated isothermal amplification

A colorimetric loop-mediated isothermal amplification (LAMP) assay with hydroxy naphthol blue was designed to amplify a region in the outer membrane lipoprotein (oprL) gene of Pseudomonas aeruginosa. The LAMP assay showed 100% specificity for the serogroup and other bacteria, and the sensitivity was 10-fold higher than that of the PCR assays. The LAMP assay could detect P. aeruginosa inoculated in mouse feces at 130 colony-forming units (CFU)/0.1 g feces (3.25 CFU/reaction). The assay was completed within 2 h from DNA extraction. In a field trial, the LAMP assay revealed that none of the 27 samples was obtained from 2 specific pathogen-free (SPF) mouse facilities that were monitoring infection with P. aeruginosa; 1 out of 12 samples from an SPF mouse facility that was not monitoring infection with P. aeruginosa and 2 out of 7 samples from a conventional mouse facility were positive for P. aeruginosa. In contrast, P. aeruginosa was not detected in any of the samples by a conventional culture assay. Thus, this colorimetric LAMP assay is a simple and rapid method for P. aeruginosa detection.     

Rapid and simple detection of eight major periodontal pathogens by the loop-mediated isothermal amplification method

Loop-mediated isothermal amplification (LAMP) was applied to develop a rapid and simple detection system for eight periodontal pathogens: Aggregatibacter (Actinobacillus) actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola and Tannerella forsythia. Primers were designed from the 16S ribosomal RNA gene for each pathogen, and the LAMP amplified the targets specifically and efficiently under isothermal condition at 64 degrees C. To simplify the manipulation of LAMP examination, boiled cells and intact cells suspended in phosphate-buffered saline (PBS) were tested as templates besides extracted DNA template. The detection limits were 1-10 cells per tube using extracted DNA template. However, LAMP methods using boiled cells and intact cells required 10-100 and 100-1000 cells per tube, respectively. LAMPs for A. actinomycetemcomitans, P. gingivalis and P. intermedia were then applied to clinical plaque samples, and the method demonstrated equal or higher sensitivity compared with the conventional real-time PCR method. These findings suggest the usefulness of the LAMP method for the rapid and simple microbiological diagnosis of periodontitis, and the possibility of LAMP examination without the DNA extraction step.