Detection of Legionella pneumophila using a real-time PCR hybridization assay

A real-time PCR hybridization assay for Legionella pneumophila is described; the assay uses LightCycler (Idaho Technology) methodology to specifically detect 2.5 CFU/reaction, equivalent to 1,000 CFU/liter of starting water sample. The assay, including DNA extraction and confirmation of product identity, is completed within 90 min of receipt of a sample.     

实时荧光PCR快速检测嗜肺军团菌的研究

目的 建立TaqMan-MGB探针实时荧光PCR快速检测嗜肺军团菌技术,为临床和环境样品检测嗜肺军团菌提供可实用工具.方法 在对嗜肺军团菌mip序列进行分析、比较基础上,设计一对特异性引物和TaqMan-MGB探针,通过实时荧光PCR反应条件和反应体系的优化,实现对嗜肺军团菌的快速检测;用克隆到pMD-19T载体上的嗜肺军团菌mip基因阳参片段和不同菌株验证方法的敏感性和特异性.结果 当用热裂解法提取DNA,25μl的反应体系中包括上、下游引物(20μmol/L)各0.6μl,探针(20μmol/L)0.4μl,模板DNA 6.0μl,反应条件为预变95℃20 S,变性95℃10 s,退火50℃ 40 s,40个循环时,TaqMan-MGB探针实时荧光PCR技术对嗜肺军团菌mip基因阳参片段最低检测浓度为0.71拷贝/μl,其循环阈值(Ct值)与模板浓度具有极好的对应关系(r=0.999);1株嗜肺军团菌标准株、12株嗜肺军团菌分离株的Ct值在13.23～16.04之间,而包括金黄葡萄球菌、鼠伤寒沙门菌、副溶血性弧菌、大肠埃希菌、铜绿假单胞菌、痢疾志贺菌共计76株其他菌PCR Ct值均大于30;整个检测过程仅需1.5 h.结论 TaqMan-MGB探针的嗜肺军团菌实时荧光PCR检测方法具有特异性和敏感性、易操作、结果准确可靠等优点,可用于嗜肺军团菌检测.     

Legionella pneumophila mip gene sequencing to investigate a cluster of pneumonia cases

AIMS: To use fluorescent antibody (FA) and PCR studies on fixed lung tissue to investigate whether Legionella pneumophila was the cause of pneumonia in a cluster of three haematology patients. METHODS: Cut sections of paraffin blocks of lung tissue were examined by direct FA (DFA) using fluorescently labelled antibody to serogroup 1 and Pontiac strains of L. pneumophila. In addition, a single tube 'hanging drop' nested PCR targeting the mip gene of Legionella was performed on DNA extracted from the lung sections. Products were sequenced using dye terminator chemistry. RESULTS: Numerous fluorescing bacteria were seen on staining with both antibodies in lung tissue from two of the patients. Identical L. pneumophila mip gene sequences were amplified from both DFA-positive lung sections. Two differing L. pneumophila mip sequences were obtained on three separate occasions from the tissue sections from the third patient negative by DFA. These sequences differed slightly from those obtained from the two DFA positive lung tissues. CONCLUSIONS: There is good epidemiological evidence to link the first two cases who had been treated in the same ward prior to development of fever within two days of each other. The significance of results is controversial for the third patient.     

Dual detection of Legionella pneumophila and Legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region

Although the majority of cases of Legionnaires’ disease (LD) are caused by Legionella pneumophila, an increasing number of other Legionella species have been reported to cause human disease. There are no clinical presentations unique to LD and hence accurate laboratory tests are required for early diagnosis. Therefore, we designed a real-time PCR assay that targets the 23S-5S rRNA intergenic spacer region (23S-5S PCR) and allows for detection of all Legionella species and discrimination of L. pneumophila from other Legionella species. In total, 271 isolates representing 50 Legionella species were tested and the assay was validated using 39 culture-positive and 110 culture-negative patient specimens collected between 1989 and 2006. PCR-positive results were obtained with all 39 culture-positive samples (100% sensitivity). Specimens that tested positive according to 23S-5S PCR, but were culture-negative, were further analysed by DNA sequencing of the amplicon or the macrophage infectivity potentiator (mip) gene. In addition to L. pneumophila, Legionella longbeachae, Legionella cincinnatiensis and Legionella micdadei were identified in the specimens. The assay showed a 7-log dynamic range displaying a sensitivity of 7.5 CFU/mL or three genome equivalents per reaction. Sixty-one specimens containing viruses or bacteria other than Legionellae were negative according to 23S-5S PCR, demonstrating its specificity. Use of this assay should contribute to the earlier detection of respiratory disease caused by Legionella species, as well as to increased rates of detection.     

Detection of mip gene by PCR for diagnosis of Legionnaires' disease.

The mip gene of Legionella pneumophila was demonstrated by PCR and probing in paired acute-phase and convalescent-phase sera from five patients with Legionnaires' disease but not in the acute-phase sera of 100 patients with pneumonia that showed no serological evidence of Legionella infection. PCR may help in cases presenting diagnostic difficulty.     

Nucleotide sequence analysis of the Legionella micdadei mip gene, encoding a 30-kilodalton analog of the Legionella pneumophila Mip protein.

After the demonstration of analogs of the Legionella pneumophila macrophage infectivity potentiator (Mip) protein in other Legionella species, the Legionella micdadei mip gene was cloned and expressed in Escherichia coli. DNA sequence analysis of the L. micdadei mip gene contained in the plasmid pBA6004 revealed a high degree of homology (71%) to the L. pneumophila mip gene, with the predicted secondary structures of the two Mip proteins following the same pattern. Southern hybridization experiments, with the plasmid pBA6004 as the probe, suggested that the mip gene of L. micdadei has extensive homology with the mip-like genes of several Legionella species. Furthermore, amino acid sequence comparisons revealed significant homology to two eukaryotic proteins with isomerase activity (FK506-binding proteins).     

Clinical Application of a Multiplex Real-Time PCR Assay for Simultaneous Detection of Legionella Species,Legionella pneumophila,and Legionella pneumophila Serogroup 1

We developed a single-tube multiplex real-time PCR assay capable of simultaneously detecting and discriminating Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1 in primary specimens. Evaluation of 21 clinical specimens and 115 clinical isolates demonstrated this assay to be a rapid, high-throughput diagnostic test with 100% specificity that may aid during legionellosis outbreaks and epidemiologic investigations.     

A real-time PCR for specific detection of the Legionella pneumophila serogroup 1 ST1 complex

ObjectiveLegionella pneumophila serogroup 1 (Lp1) sequence type (ST) 1 is globally widespread in the environment and accounts for a significant proportion of Legionella infections, including nosocomial Legionnaires' disease (LD). This study aimed to design a sensitive and specific detection method for Lp ST1 that will underpin epidemiological investigations and risk assessment.MethodsA total of 628 Lp genomes (126 ST1s) were analyzed by comparative genomics. Interrogation of more than 900 accessory genes revealed seven candidate targets for specific ST1 detection and specific primers and hydrolysis probes were designed and evaluated. The analytical sensitivity and specificity of the seven primer and probe sets were evaluated on serially diluted DNA extracted from the reference strain CIP107629 and via qPCR applied on 200 characterized isolates. The diagnostic performance of the assay was evaluated on 142 culture-proven clinical samples from LD cases and a real-life investigation of a case cluster.ResultsOf seven qPCR assays that underwent analytical validation, one PCR target (lpp1868) showed higher sensitivity and specificity for ST1 and ST1-like strains. The diagnostic performance of the assay using respiratory samples corresponded to a sensitivity of 95% (19/20) (95% CI (75.1–99.9)) and specificity of 100% (122/122) (95% CI (97–100)). The ST1 PCR assay could link two out of three culture-negative hospitalized LD cases to ST1 during a real-time investigation.ConclusionUsing whole genome sequencing (WGS) data, we developed and validated a sensitive and specific qPCR assay for the detection of Lp1 belonging to the ST1 clonal complex by amplification of the lpp1868 gene. The ST1 qPCR is expected to deliver an added value for Lp control and prevention, in conjunction with other recently developed molecular assays.     

Comparison of Sample Preparation Methods for Detection of Legionella pneumophila in Culture-Positive Bronchoalveolar Lavage Fluids by PCR

A prospective study was conducted on 25 Legionella pneumophila culture-positive and 98 culture-negative bronchoalveolar lavage fluid samples to compare two DNA preparation methods: a rapid modified Chelex-based protocol and a proteinase K method. PCR was found to be more sensitive with the Chelex-based method (P = 0.03). No difference was found concerning the inhibition rate.     

Characterization of the Legionella pneumophila gene ligA

Legionella pneumophila is a bacterial pathogen that resides and multiplies in macrophages as well as in its natural aquatic hosts, the protozoa. Different bacterial factors contribute to pathogenicity and accompanying eukaryotic intracellular events. Sequencing of mip flanking regions revealed a gene of 2610 bp, ligA, that has no significant similarity to any of the genes identified previously. Epidemiological studies indicate that this gene is present in Legionella pneumophila, the species most often associated with cases of the Legionnaires' disease, but not in Legionella species other than L. pneumophila. The isogenic ligA deletion mutant was resistant to NaCl, and showed decreased cytotoxicity to human monocytes and decreased hemolytic activity to red blood cells. However, the most prominent effect of the L. pneumophila ligA mutant strain LEPF1 was the nearly completely reduced replication within the natural host Acanthamoeba castellanii. Since this gene is L. pneumophila specific and regulates numerous bacterial properties we designated this gene ligA for Legionella pneumophila infectivity gene A.     

Direct detection and differentiation of Legionella spp. and Legionella pneumophila in clinical specimens by dual-color real-time PCR and melting curve analysis

A dual-color LightCycler PCR assay targeting the 16S rDNA gene of Legionella spp. was established. By using two pairs of hybridization probes, Legionella spp. and Legionella pneumophila could be detected and differentiated simultaneously. With 26 culture-positive and 42 culture-negative respiratory specimens from patients with atypical pneumonia, 100% sensitivity and specificity was observed for L. pneumophila.     

Development and clinical evaluation of an internally controlled,single-tube multiplex real-time PCR assay for detection of Legionella pneumophila and other Legionella species

A multiplex real-time PCR assay for detection of Legionella pneumophila and Legionella spp. and including an internal control was designed. Legionella species, L. pneumophila, and the internal control were detected simultaneously by probes labeled with 6-carboxy-fluorescein, hexachlorofluorescein, and indodicarbocyanine, respectively. Therefore, no postamplification analysis was required in order to distinguish the targets. The sensitivity of both assays was 2.5 CFU/ml, and from analysis of 10 culture-positive and 74 culture-negative samples from patients investigated for legionellosis, 100% agreement was observed by both assays in comparison to culture. Four additional positives were found by the multiplex real-time PCR assay in the Legionella culture-negative samples.     

Detection of Legionella pneumophila capsular-like envelope antigens by counterimmunoelectrophoresis.

The capsular-like envelope of Legionella pneumophila strains Togus 1 (serotype 2) and Philadelphia 1 (serotype 1) was isolated and purified by column chromatography on Sepharose 6B. Antibody raised in rabbits to these two antigenic materials did not cross-react in gel diffusion. Upon electrophoresis followed by gel diffusion, the majority of both envelope materials was found to migrate towards the cathode. A minor antigenic component of each envelope only migrated slightly towards the anode. Using the envelope antigens and the two anti-envelope sera in a counterimmunoelectrophoresis (CIE) assay, positive results were only obtained when the antigenic materials were placed in the cathodal well. The Togus 1 and Philadelphia 1 antigens did not cross-react in CIE. The sensitivity of the CIE assay was poor (15.6 micrograms/ml by carbohydrate content) compared to its sensitivity in other microbial systems. Although CIE may not be a useful diagnostic aid in identifying Legionella species due to its low sensitivity, it may be of value in serotyping the microorganism since we did not see cross-reactivity between the two strains when anti-envelope sera were used.     

The occurrence of Legionella species other than Legionella pneumophila in clinical and environmental samples in Denmark identified by mip gene sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry

In Denmark, several laboratories use PCR as a routine diagnostic method for Legionnaires' disease, and almost all PCR-positive samples are investigated by culture. From 1993 to 2010, isolates of Legionella species other than Legionella pneumophila were obtained from respiratory samples from 33 patients, and from 1997 to 2010, 42 isolates of Legionella non-pneumophila species were obtained and saved from water samples from 39 different sites in Denmark. Macrophage infectivity potentiator gene (mip) sequencing was used as a reference method to identify the Legionella non-pneumophila species. Only one of the 75 isolates did not meet the acceptance criterion of a similarity of ≥98% to sequences in the database. The species distribution between clinical and environmental isolates varied. For the former, four species were detected, with Legionella bozemanae and Legionella micdadei predominating (both 44%). For the latter, eight species were detected, with Legionella anisa predominating (52%). The distribution among the Danish clinical isolates was different from the general distribution both in Europe and outside Europe, where L. bozemanae and Legionella longbeachae are the most commonly found clinical Legionella non-pneumophila species. The 75 isolates were also investigated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): 64 were correctly identified, with a score of ≥2.0; eight had a score of <2.0, but only two of these were wrongly identified; and three gave no results with MALDI-TOF MS. Both mip sequencing and MALDI-TOF MS are robust methods for Legionella species identification.     

Evaluation of a real-time PCR hybridization assay for rapid detection of Legionella pneumophila in hospital and environmental water samples

A real-time LightCycler assay for Legionella pneumophila was evaluated with 120 water samples potentially contaminated with PCR inhibitors. Results were obtained within five hours, with a detection limit equivalent to 800 cells/L. However, 11 of 22 culture-positive samples containing < 100 CFU/L were also positive by LightCycler assay, indicating the presence of significant numbers of non-viable cells. Following extraction, amplification inhibitors remained in four culture-positive samples, but only one contained > 800 CFU/L. The assay seemed suitable for rapidly screening large sample numbers for heavy contamination with L. pneumophila , but conventional culture should continue to be used to detect low contamination levels.     

Molecular typing of nosocomial strains of Legionella pneumophila by arbitrarily primed PCR.

Arbitrarily primed PCR with two different primers was compared with ribotyping and monoclonal antibody analysis for typing Legionella strains. Applied to 11 epidemiologically unrelated strains, arbitrarily primed PCR resulted in an index of discrimination of 100% with both primers. It was found able to identify an epidemic clone of Legionella pneumophila serogroup 1 that was isolated from both patients and a hot water circuit of the same hospital.     

Sequence-Based Classification Scheme for the Genus Legionella Targeting the mip Gene

The identification and speciation of strains of Legionella is often difficult, and even the more successful chromatographic classification techniques have struggled to discriminate newly described species. A sequence-based genotypic classification scheme is reported, targeting approximately 700 nucleotide bases of the mip gene and utilizing gene amplification and direct amplicon sequencing. With the exception of Legionella geestiana, for which an amplicon was not produced, the scheme clearly and unambiguously discriminated among the remaining 39 Legionella species and correctly grouped 26 additional serogroup and reference strains within those species. Additionally, the genotypic classification of approximately 150 wild strains from several continents was consistent with their phenotypic classification, with the exception of a few strains where serological cross-reactivity was complex, potentially confusing the latter classification. Strains thought to represent currently uncharacterized species were also found to be genotypically unique. The scheme is technically simple for a laboratory with even basic molecular capabilities and equipment, if access to a sequencing laboratory is available.The genus Legionella comprises approximately 40 species, at least 7 of which have more than one serotype (3, 15, 31). Approximately half of the species have been associated with human disease (28). Legionella-like organisms isolated from clinical specimens, or from the environment during the course of an outbreak, need to be identified to elucidate the disease process and to identify the source. Legionellae have proved to be relatively unreactive when traditional biochemical tests are utilized, necessitating more complex identification methods (6, 7, 26, 41). Serologically based methods are widely used in clinical laboratories, but antigen cross-reactivity limits specificity and restricts their confident use to a few frequently isolated species (38). This is especially true for countries where legionellosis caused by species other than L. pneumophila is common (12). More complex classification schemes have been proposed (26, 38), the most successful being one based on the range and proportion of cellular fatty acids and ubiquinones (21, 22, 40, 43). As additional species have been characterized, this method has become less discriminating, since apparently unique patterns were proved to be shared by several species (43). The inclusion of hydroxylated fatty acids has improved discrimination, but it requires the analysis of both mono- and dihydroxylated fatty acids, and individual patterns are complex, making analysis difficult (21).Gene sequence-based phylogenic (genotypic) schemes have become widely used for organisms which are difficult to classify, as more sequences have been determined and sequencing methods have become simpler, more widely available, and cost effective (11, 23, 24, 29, 32, 34). Genotypic schemes have the great advantage of being unaffected by colony age and growth conditions and, in contrast to chromatographic methods, are not subject to extraction and chromatographic conditions or constituent equipment. Additionally, because a gene sequence is essentially a long digital string, with each digit being one of only four nucleotides, genotypic schemes are less ambiguous and can utilize significantly more discriminatory data than phenotypic ones, and in a form that lends itself to widely available computer analysis software. Many genotypic schemes utilize variation in the 16S rRNA sequence (11, 23, 24, 32, 34), because of the ease with which regions can be amplified and sequenced with universal primers. The 16S rRNA sequences of Legionella species have been reported (18), as have the sequences of the mip gene (2, 12, 13, 31), which codes for an immunophilin of the FK506 binding protein (FKBP) class (14). This protein, which ranges in size from 232 to 251 amino acids, depending on the Legionella species (31), is an outer membrane protein important in the intracellular cycle of Legionella. While it is known to be involved with the survival of the bacterium immediately after uptake into phagocytic cells (9, 12, 28), its exact role is unclear. Additionally, analogs are found widely in both prokaryotes and eukaryotes and are likely to have a significant cellular role (14). Other gene sequences have been determined for Legionella (5, 17, 36), but only the rRNA sequences and the mip gene have been comprehensively determined for most species, an essential prerequisite for any gene to be the basis of a genotyping scheme. Ratcliff et al. (31) recently phylogenetically compared most Legionella species, using the species variation among both the 16S rRNA and mip genes, and found over twice the variation in the mip gene at the DNA level (56% of base sites) as in 16S rRNA (23% of base sites). A pairwise comparison of species reveals a mip gene variation of 3 to 31% (mean, 20%) between species pairs compared with 1 to 10% (mean, 6%) for 16S rRNA. For the mip gene, interspecies nucleotide variation occurred throughout the gene but especially within a hypervariable insert of up to 51 bases immediately adjacent to the region coding for the signal sequence, at redundant third codon sites, and in sequences coding for either single or small regions of variable amino acids interspersed among regions coding for total or near-total amino acid homology, especially toward the 3′ end of the gene (31). These last regions are known to encode the active portions of the protein’s enzymatic peptidylprolyl cis-trans-isomerase (PPIase) activity (31).Additionally the mip gene appeared to be relatively stable genetically, with no evidence of homologous recombination, in that identical or near-identical sequences were not found for the mip genes from phenotypically divergent species. With respect to genetic stability, the mip gene may therefore behave like housekeeping genes, which are known to be more stable than other gene classes (1). Homologous recombination would severely compromise a sequence-based classification scheme (1), and it is a theoretical possibility at least for rRNA targets (37). Thus, the genetic stability and greater mutational variation of the mip gene suggest that it is an ideal target for a classification scheme, with results likely to be more discriminating in identifying species and more resilient to clonal variation within each species. It may even be possible to discriminate between serogroups where these are present or to demonstrate distinct intraspecies clonal groups.The present study reports the use of the mip gene to develop a sequence-based classification scheme for Legionella, the first proposed for this genus. Further, it reports the comparison of sequences from species which have additional serogroups, to determine if serogroups can be discriminated. Similarly, it reports the comparison of sequences from wild strains isolated on several continents, for which there is confirmatory phenotypic or DNA hybridization identification data, to test the robustness of the scheme for variation within strains of the same species. Lastly, isolates which appear phenotypically or from DNA hybridization studies to be different from currently characterized species were tested to determine if a sequence-based classification scheme can clarify their identities. Some of these unusual isolates have been previously reported (43).     

实时荧光定量PCR检测沙眼衣原体的研究

目的采用TaqMan探针建立检测沙眼衣原体的实时荧光定量PCR（real-time PCR）方法。方法根据沙眼衣原体外膜蛋白A的基因（ompA）序列设计引物和探针,以克隆的ompA部分基因片段作DNA模板,建立实时荧光定量检测方法。结果建立的荧光定量PCR检测方法的最低检出限为5 copies/反应,检测线性范围100～107线性关系良好（r2=0.997）,比巢式PCR敏感100倍;且与鹦鹉热衣原体、淋球菌、解脲脲原体、大肠杆菌等病原菌DNA以及人基因组DNA均无交叉反应,表明该方法具有良好的特异性。以巢式PCR作参比,建立的荧光定量PCR法检测沙眼衣原体的阳性符合率为100.00%,阴性符合率为95.09%,总符合率为96.81%。结论建立的检测沙眼衣原体实时荧光定量PCR具有特异性强和敏感性高的特点,可快速检测样本中微量沙眼衣原体DNA,适用于对沙眼衣原体进行大规模筛选。