Characterization of Bordetella holmesii isolates from patients with pertussis-like illness in The Netherlands

Bordetella holmesii is a recently described human pathogen mainly isolated from blood. However, in the US and Canada, B. holmesii has also been cultured from the nasopharynx of patients with pertussis-like symptoms. To the best of our knowledge, respiratory isolates from Europe have not been characterized. Here, we report the isolation and characterization of B. holmesii from Dutch patients with pertussis-like illness. Species determination was confirmed by 16S rRNA gene sequencing and detection by PCR of IS481 and bhoE, a gene not found in Bordetella pertussis but present in B. holmesii. Comparative genomic hybridization (CGH) with microarrays revealed that the Dutch isolates formed a cluster distinct from isolates from the US and UK suggesting a distinct population or an epidemiological relationship between the Dutch isolates. All isolates contained a locus involved in iron uptake, previously suggested to originate from B. pertussis. The causes for the apparent increase in the isolation of B. holmesii are discussed.     

Characterization of a highly conserved island in the otherwise divergent Bordetella holmesii and Bordetella pertussis genomes

The recently discovered pathogen Bordetella holmesii has been isolated from the airways and blood of diseased humans. Genetic events contributing to the emergence of B. holmesii are not understood, and its phylogenetic position among the bordetellae remains unclear. To address these questions, B. holmesii strains were analyzed by comparative genomic hybridization (CGH) to a Bordetella pertussis microarray and by multilocus sequence typing. Both methods indicated substantial sequence divergence between B. pertussis and B. holmesii. However, CGH identified a putative pathogenicity island of 66 kb that is highly conserved between these species and contains several IS481 elements that may have been laterally transferred from B. pertussis to B. holmesii. This island contains, among other genes, a functional, iron-regulated locus encoding the biosynthesis, export, and uptake of the siderophore alcaligin. The acquisition of this genomic island by B. holmesii may have significantly contributed to its emergence as a human pathogen. Horizontal gene transfer between B. pertussis and B. holmesii may also explain the unusually high sequence identity of their 16S rRNA genes.     

Molecular characterization of the BvgA response regulator of Bordetella holmesii

The BvgAS system controls the expression of most virulence factors in Bordetella pertussis. Recently, we identified an orthologous system in the related human pathogen Bordetella holmesii. However, while we found that the orthologous histidine kinases BvgS could be functionally exchanged between the two species, the B. holmesii response regulator BvgA(BH) could not substitute for its B. pertussis counterpart in vivo and, accordingly, was not able to bind to B. pertussis virulence promoters in vitro. Here we show that a hybrid response regulator consisting of the B. pertussis derived DNA-binding output domain of BvgA(BP) combined with the B. holmesii receiver domain binds to BvgA(BP) regulated virulence promoters of B. pertussis in vitro and is functional in B. pertussis in vivo. This shows that the inability of BvgA(BH) to complement BvgA(BP) in B. pertussis is due to the small number of sequence variations present in its output domain. However, by mutation analysis we show that four amino acid exchanges present in the helix-turn-helix motif of BvgA(BH) as compared to BvgA(BP) are not the only reason for its inability to substitute for BvgA(BP) but additional mutations present in the output domain must play a role.     

Genetic diversity analysis of isolates belonging to Bordetella genus

In this study, RandomAmplified Polymorphic DNA (RAPD) method was used to track differences among human and animals isolates of eight known species of Bordetella genus. One hundred representative strains of these species from international and Polish bacterial collections were genotyped according to RAPD protocols using primer 1254 or 1247. Problems with reproducibility and discriminatory power, frequently discussed in literature, have been overcome by precise optimization procedure, which allowed to achieve reliable conditions for Bordetella species analysed. This study proved high potential of RAPD method using primers 1247/1254 for intra-species differentiation of B. bronchiseptica and B. hinzii isolates. Additionally RAPD method has been found to be useful for confirming B. avium and B. holmesii identification.     

A simple and sensitive method for detection of Bacillus anthracis by loop‐mediated isothermal amplification

Aims: To develop a rapid and simple system for detection of Bacillus anthracis using a loop‐mediated isothermal amplification (LAMP) method and determine the suitability of LAMP for rapid identification of B. anthracis infection. Methods and Results: A specific LAMP assay targeting unique gene sequences in the bacterial chromosome and two virulence plasmids, pXO1 and pXO2, was designed. With this assay, it was possible to detect more than 10 fg of bacterial DNA per reaction and obtain results within 30–40 min under isothermal conditions at 63°C. No cross‐reactivity was observed among Bacillus cereus group and other Bacillus species. Furthermore, in tests using blood specimens from mice inoculated intranasally with B. anthracis spores, the sensitivity of the LAMP assay following DNA extraction methods using a Qiagen DNeasy kit or boiling protocol was examined. Samples prepared by both methods showed almost equivalent sensitivities in LAMP assay. The detection limit was 3·6 CFU per test. Conclusions: The LAMP assay is a simple, rapid and sensitive method for detecting B. anthracis. Significance and Impact of the Study: The LAMP assay combined with boiling extraction could be used as a simple diagnostic method for identification of B. anthracis infection.     

Loop-mediated isothermal amplification for rapid detection of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> spores

A loop-mediated isothermal amplification (LAMP) assay system was employed for detecting Bacillus anthracis spores in pure cultures as well as in various simulated powder samples. The specificity of the designed LAMP primer sets was validated by assaying 13 B. anthracis strains and 33 non-B. anthracis species. The detection limits of the LAMP assay were 10 spores/tube for pure cultures and 100 spores/2 mg powder for simulated powder samples. The results show that the LAMP protocol is a promising method for detecting B. anthracis.     

Rapid and simple detection of Clostridium botulinum types A and B by loop-mediated isothermal amplification

Aims:  To develop a convenient and rapid detection method for toxigenic Clostridium botulinum types A and B using a loop-mediated isothermal amplification (LAMP) method.
Methods and results:  The LAMP primer sets for the type A or B botulinum neurotoxin gene, BoNT / A or BoNT / B , were designed. To determine the specificity of the LAMP assay, a total of 14 C. botulinum strains and 17 other Clostridium strains were tested. The assays for the BoNT/A or BoNT/B gene detected only type A or B C. botulinum strains, respectively, but not other types of C. botulinum or strains of other Clostridium species. Using purified chromosomal DNA, the sensitivity of LAMP for the BoNT/A or BoNT/B gene was 1 pg or 10 pg of DNA per assay, respectively. The assay times needed to detect 1 ng of DNA were only 23 and 22 min for types A and B, respectively. In food samples, the detection limit per reaction was one cell for type A and 10 cells for type B.
Conclusions:  The LAMP is a sensitive, specific and rapid detection method for C. botulinum types A and B.
Significance and Impact of the Study:  The LAMP assay would be useful for detection of C. botulinum in environmental samples.     

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.     

Identification and characterization of a brilliant yellow pigment produced by Bordetella pertussis

Culture supernatants of Bordetella pertussis are a brilliant yellow; however, the structure and biological role of the responsible pigment have not been investigated. In this study, a brilliant yellow‐colored fraction was extracted from culture supernatants of B. pertussis and analyzed by HPLC. UV–visible spectral analysis and mass spectrometry identified the brilliant yellow pigment as riboflavin. Riboflavin production was high in lag and early log phases and riboflavin was found to enhance growth of B. pertussis in low‐density cultures. Riboflavin production is not regulated by the BvgAS system. In addition, it was found that other Bordetella species, such as B. parapertussis , B. holmesii and B. bronchiseptica, also release riboflavin into their culture supernatants. This is the first report that B. pertussis secrets riboflavin to the extracellular space and that riboflavin may promote its growth. The mechanism may be associated with pathogenesis of B. pertussis .

     

用于高灵敏可视化检测松材线虫的闭管等温扩增法

建立了一种基于环介导等温核酸扩增技术(LAMP)的松材线虫高灵敏可视化闭管检测方法。针对松材线虫核糖体DNA的序列保守区域设计LAMP引物,通过优化LAMP体系中的Mg2+、甜菜碱浓度和反应温度等因素,建立了环介导等温扩增法;并结合蜡封反应管对产物进行检测,检测结果可直接通过肉眼观察SYBR Green I荧光显色进行判定。结果表明,本方法可检测到低至10拷贝/管的松材线虫核酸片段,可对单条线虫进行检测,并且具有很高的特异性,能区分检测松材线虫与拟松材线虫。由于整个反应恒温进行,无需热循环仪;闭管检测极大地降低了扩增产物交叉污染的风险;检测速度快,整个检测过程只需40 min,为松材线虫的现场快速筛检提供了一种简便、高灵敏、高特异的工具。     

Rapid and Sensitive Detection of Bartonella bacilliformis in Experimentally Infected Sand Flies by Loop-Mediated Isothermal Amplification (LAMP) of the Pap31 Gene

Background

Carrion'' disease, caused by Bartonella bacilliformis, remains truly neglected due to its focal geographical nature. A wide spectrum of clinical manifestations, including asymptomatic bacteremia, and lack of a sensitive diagnostic test can potentially lead to a spread of the disease into non-endemic regions where competent sand fly vectors may be present. A reliable test capable of detecting B. bacilliformis is urgently needed. Our objective is to develop a loop-mediated isothermal amplification (LAMP) assay targeting the pap31 gene to detect B. bacilliformis.

Methods and Findings

The sensitivity of the LAMP was evaluated in comparison to qPCR using plasmid DNA containing the target gene and genomic DNA in the absence and presence of human or sand fly DNA. The detection limit of LAMP was 1 to 10 copies/µL, depending on the sample metrics. No cross-reaction was observed when testing against a panel of various closely related bacteria. The utility of the LAMP was further compared to qPCR by the examination of 74 Lutzomyia longipalpis sand flies artificially fed on blood spiked with B. bacilliformis and harvested at days (D) 1, 3, 5, 7 and 9 post feeding. Only 86% of sand flies at D1 and 63% of flies at D3 were positive by qPCR. LAMP was able to detect B. bacilliformis in all those flies confirmed positive by qPCR. However, none of the flies after D3 were positive by either LAMP or qPCR. In addition to demonstrating the sensitivity of the LAMP assay, these results suggest that B. bacilliformis cannot propagate in artificially fed L. longipalpis.

Conclusions

The LAMP assay is as sensitive as qPCR for the detection of B. bacilliformis and could be useful to support diagnosis of patients in low-resource settings and also to identify B. bacilliformis in the sand fly vector.     

Loop-mediated isothermal amplification for the rapid detection of Haemophilus parasuis

Haemophilus parasuis infection is of considerable economic importance in the swine industry due to the high costs associated with treatment and loss of animals all over the world. In the present study, loop-mediated isothermal amplification (LAMP) is described for the rapid and specific detection of this species. A primer set derived from the inf B gene of H. parasuis was used to validate the assay using 15 H. parasuis reference strains, 39 clinical isolates, 75 positive samples, and 18 other pathogens. The results indicated that positive reactions were confirmed for all H. parasuis strains and specimens by LAMP after 45 min reaction at 65 °C in a water bath, and no cross-reactivity was observed from other non-H. parasuis strains. The detection limit of the conventional PCR was 25 copies, while that of the LAMP was five copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. LAMP is likely to be more suitable as a routine diagnostic tool, especially in clinics without complicated equipment such as thermal cycling machines and electrophoresis apparatus. In these scenarios, the H. parasuis LAMP assay has the potential for field diagnosis.     

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。     

Loop-mediated isothermal amplification for the rapid detection of Salmonella

Loop-mediated isothermal amplification (LAMP) assay detected Salmonella within 60 min. The 220 strains of 39 serotypes of Salmonella subsp. enterica and 7 strains of Salmonella enterica subsp. arizonae were amplified, but not 62 strains of 23 bacterial species other than Salmonella. The sensitivity of the LAMP assay was found to be >2.2 cfu/test tube using nine serotypes. The specificity was similar to that of a PCR assay, but the sensitivity of LAMP was greater. Both fluorescence and turbidity were able to detect the products in the LAMP assay. S. enteritidis in a liquid egg sample artificially inoculated with the organism was detected by the LAMP assay at 2.8 cfu/test tube, although negative by PCR assay. These results indicate that the LAMP assay is a rapid, specific and sensitive detection method for Salmonella.     

环介导恒温扩增对莱姆病病原伯氏疏螺旋体的分型鉴定

使用环介导恒温扩增技术,基于莱姆病病原伯氏疏螺旋体的外膜蛋白A(OspA)基因,针对伯氏疏螺旋体不同的基因型设计特异性引物,对国内主要的莱姆病病原伯氏疏螺旋体的3个基因型进行分型鉴定。研究结果表明,设计的引物具有良好的特异性,可以对狭义伯氏疏螺旋体(Borrelia burgdorferi sensu strict)、嘎氏疏螺旋体(B.afzelii)和伽氏疏螺旋体(B.garinii)进行分型鉴定。伯氏疏螺旋体的分型鉴定可以对不同临床症状莱姆病患者的治疗和莱姆病的控制提供一定的依据。     

Development and Evaluation of a Novel and Rapid Detection Assay for Botrytis cinerea Based on Loop-Mediated Isothermal Amplification

Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63°C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10⁻³ ng µL⁻¹ of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10⁻² ng µL⁻¹). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables.     

Development and evaluation of a loop‐mediated isothermal amplification assay for rapid detection of chicken anaemia virus

Aim: Chicken anaemia virus (CAV) causes an economically important viral disease in chickens worldwide. The main aim of this study was to establish a rapid, sensitive and specific loop‐mediated isothermal amplification (LAMP) assay for detecting CAV infection. Methods and Results: A set of four specific LAMP primers were designed based on the nucleotide sequence of the CAV VP2 gene, which encodes a nonstructural protein. These were used for the amplification of a specific target region of the VP2 gene. LAMP amplicons were successfully amplified and detected by DNA electrophoresis and by direct naked eye SYBR Green I visualization. A sensitivity test systematically demonstrated that the LAMP assay was superior to a conventional PCR assay with a minimum concentration limit of 100 fg compared to 10 ng for the conventional PCR. The specificity of the LAMP assay for CAV detection is consistent with conventional PCR. Using this established LAMP assay, infected and uninfected clinical samples obtained from an experimental farm were fully verified. Conclusions: A novel nucleic acid‐based approach of LAMP assay was successfully developed for detecting CAV infection. Significance and Impact of the Study: In this study, these results indicate that the developed LAMP assay herein for CAV detection is a time‐effective, simple, sensitive and specific test that can be used as an alternative approach in the future for large‐scaled diagnosis on the farm of CAV infection.     

用环介导等温扩增技术快速检测粪便样本中的沙门菌

目的:建立快速检测粪便样本中的沙门菌的环介导等温扩增技术(LAMP),并着重在灵敏度和特异性方面对此方法进行评价。方法:利用LAMP针对沙门菌特定基因invA(靶基因)设计的6条特异引物,通过引物特异性识别特定基因invA上的8个独立区域来快速检测沙门菌;LAMP反应过程中会产生白色沉淀焦磷酸镁,故可以通过监测浊度来判定反应结果。结果:实时浊度仪监测反应结果表明,LAMP反应在60~65℃等温条件下50 min内完成;如果在反应前添加羟基萘酚兰,蓝色阳性结果很明显区别于紫色阴性结果;LAMP的最低检出限为6.97 pg/μL,PCR为69.7pg/μL,LAMP方法的检测灵敏度是PCR的10倍,且具有良好的特异性。结论:LAMP方法用于快速检测沙门菌,具有检测过程简单、实验装置简便、反应结果肉眼可辨、灵敏度高、特异性强的特点,对非沙门菌菌株的结果呈阴性,表明引物设计有很好的特异性。对粪便样本进行检测,发现具有同样的敏感性和特异性。这表明LAMP法是潜在的和有价值的在粪便样本中直接检测沙门菌的方法,具有快速、简便、低成本的特点。LAMP法适用于快速临床诊断。