Detection and quantification of Campylobacter jejuni and Campylobacter coli using real-time multiplex PCR

Aims: We describe a real‐time quantitative multiplex polymerase chain reaction (qmPCR) assay to identify and discriminate between isolates of Campylobacter jejuni and Campylobacter coli. Methods and Results: Two novel sets of primers and hydrolysis probes were designed to amplify the unique DNA sequences within the hipO, ccoN and cadF genes that are specific to Camp. jejuni and Camp. coli. Using the designed optimized qmPCR assay conditions, the amplification efficiency is in range from 108 to 116%. These qmPCR assays are highly specific for Camp. jejuni and Camp. coli, as seen through testing of 40 Campylobacter strains and 17 non‐Campylobacter strains. In chicken juice and tap water models spiked with known quantities of Camp. jejuni, qmPCR detected 10²–10³ CFU ml^?1 within 4 h. Conclusions: The qmPCR assays developed in this study provide reliable and simultaneous detection and quantification of Camp. jejuni and Camp. coli, with good amplification reaction parameters. Significance and Impact of the Study: Following further validation, the qmPCR assay reported here has the potential to be applied to various sample types as an alternative and rapid methodology.     

Development of a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection and identification of Campylobacter jejuni, Campylobacter coli and Campylobacter fetus

A cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection of cdtA, cdtB or cdtC gene of Campylobacter jejuni, Campylobacter coli or Campylobacter fetus, respectively, was developed and evaluated with 76 Campylobacter strains belonging to seven different species and 131 other bacterial strains of eight different genera. The cdtA, cdtB or cdtC gene of C. jejuni, C. coli or C. fetus, respectively, could be successfully amplified using the corresponding set of primers in a highly species-specific manner. Furthermore, the specific primer set for the cdtA, cdtB or cdtC gene of a particular species could amplify the desired gene from a mixture of DNA templates of any of two or all three species. The detection limit of C. jejuni, C. coli or C. fetus was 10-100 CFU tube(-1) by the multiplex PCR assay on the basis of the presence of the cdtA, cdtB or cdtC gene. These data indicate that the cdt gene-based multiplex PCR assay may be useful for rapid and accurate detection as well as identification of Campylobacter strains in a species-specific manner.     

Routine identification of Campylobacter jejuni and Campylobacter coli from human stool samples

Correct identification of Campylobacter jejuni and Campylobacter coli isolates to the species or subspecies level is a cumbersome but nevertheless important task for a routine diagnostic laboratory. The widely used biochemical tests might be often misleading while more sophisticated phenotypic or genotypic methods are not generally available. This investigation was performed to assess the performance of common biochemical identification in comparison with species-specific PCR and gas liquid chromatography of whole cell fatty acid extracts (GLC). A total of 150 consecutive isolates from human stool samples were investigated (134 C. jejuni ssp. jejuni, 14 C. coli, two Helicobacter pullorum). From these 144, 145 and 149 isolates were correctly identified by biochemistry, GLC and PCR, respectively. Biochemical identification of all C. jejuni isolates was confirmed by PCR. GLC detected both H. pullorum strains but misidentified two C. coli strains as C. jejuni and one C. jejuni strain as C. coli. No single method can be defined as 'gold standard' for identification of C. jejuni and C. coli but a combination of techniques is needed. Therefore a stepwise identification scheme starting with biochemical reactions is suggested. All results other than C. jejuni should be confirmed by further methods. For indoxyl acetate-positive isolates species-specific PCR is recommended while GLC seems to be advantageous in indoxyl acetate-negative isolates.     

MALDI-TOF-MS方法快速鉴定食品中空肠弯曲菌的技术研究

运用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of-flight mass spectrometry,MALDI-TOF-MS)技术快速鉴定食品中空肠弯曲菌.通过对该方法的样品前处理的选择、稳定性、特异性等方面进行研究,确定了方法...     

Application of real-time PCR for quantitative detection of Campylobacter jejuni in poultry, milk and environmental water

Campylobacter jejuni is a leading human food-borne pathogen. The rapid and sensitive detection of C. jejuni is necessary for the maintenance of a safe food/water supply. In this article, we present a real-time polymerase chain reaction (PCR) assay for quantitative detection of C. jejuni in naturally contaminated poultry, milk and environmental samples without an enrichment step. The whole assay can be completed in 60 min with a detection limit of approximately 1 CFU. The standard curve correlation coefficient for the threshold cycle versus the copy number of initial C. jejuni cells was 0.988. To test the PCR system, a set of 300 frozen chicken meat samples, 300 milk samples and 300 water samples were screened for the presence of C. jejuni. 30.6% (92/300) of chicken meat samples, 27.3% (82/300) of milk samples, and 13.6% (41/300) of water samples tested positive for C. jejuni. This result indicated that the real-time PCR assay provides a specific, sensitive and rapid method for quantitative detection of C. jejuni. Moreover, it is concluded that retail chicken meat, raw milk and environmental water are commonly contaminated with C. jejuni and could serve as a potential risk for consumers in eastern China, especially if proper hygienic and cooking conditions are not maintained.     

空肠弯曲菌的磁捕获_-荧光PCR检测方法的建立

为提高畜禽类食品中空肠弯曲菌的检出率和灵敏度,应用抗血清和磁性微珠首次制备弯曲菌免疫磁珠,利用弯曲菌免疫磁珠直接捕获检样中目的菌,不需要增菌培养;通过荧光PCR检测鞭毛蛋白A(flaA)基因和/或马尿酸酶(hipO)基因,首次建立空肠弯曲菌的磁捕获-荧光聚合酶链反应(IMC-FPCR)方法.IMC-FPCR法检测空肠弯曲菌方法简便易行,可在24h内完成,特异性好,检测低限达到10cfu/mL,抗干扰性强.IMC-FPCR方法可望解决非可培养状态的空肠弯曲菌检测难题,是一种适用于检验检疫、卫生防疫和农产品安全检验等领域的快速方法.     

Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory

Aims:  To facilitate efficient identification of commonly encountered mycobacteria species ( Mycobacterium tuberculosis , Mycobacterium avium , Mycobacterium intracellulare , Mycobacterium fortuitum complex , Mycobacterium chelonae/abscessus , Mycobacterium kansasii , Mycobacterium gordonae ) in high throughput laboratories, a 16s rDNA sequence based real-time PCR assay was developed and evaluated.
Methods and Results:  Oligonucleotide primers and hybridization probes were designed based on sequence differences of the mycobacterial 16S rDNA gene. This assay was evaluated with 1649 suspected non-tuberculosis mycobacterial isolates. Apart from 3 out of 40  M. avium isolates that showed false signal with M. intracellulare specific probe, 100% specificity was obtained for all tested probes. Assay sensitivity varied from 88·9 to 100% depending on species. Average cost for obtaining a definite identification was only USD 1·1 with an average turn around time of less than 3 days.
Conclusions:  A rapid, simple and inexpensive real-time PCR assay was developed for the identification of common encountered mycobacteria in a high throughput laboratory setting.
Significance and Impact of the Study:  With this assay, more than 80% of the clinically isolated nontuberculous mycobacteria could be identified in a highly cost effective manner. This helped to save resources for other laboratory activities especially in high throughput mycobacterial laboratories.     

Shedding of Campylobacter spp. in Finnish cattle on dairy farms

Aims:  The aim of this study was to determine variation of prevalence throughout a year, colonization levels and genotypes of Campylobacter jejuni in Finnish dairy cattle herds.
Methods and Results:  Faecal samples and tank milk samples from three dairy cattle herds were taken five times, and swab samples from drinking troughs once during a 1-year sampling period. The samples were enriched in Bolton broth and subsequently spread on mCCDA. Isolates were then subtyped by pulsed-field gel electrophoresis using SmaI. Campylobacter jejuni was detected in 169 of the 340 faecal samples and in one drinking trough sample. Prevalences between herds and sampling times varied widely. The faecal levels of C. jejuni were mainly low. Between one and four SmaI subtypes were identified from each herd per sampling. Two SmaI subtypes persisted in two of the herds throughout the study.
Conclusions:  Dairy cattle can be a long-term reservoir of C. jejuni subtypes similar to clinical isolates. Differences in the colonization potential among C. jejuni strains as well as in the resistance to campylobacter colonization among animals are possible.
Significance and Impact of the Study:  The study provides data on contamination dynamics, colonization levels and the persistence of C. jejuni in dairy cattle.     

Development of a PCR assay combined with a short enrichment culture for detection of Campylobacter jejuni in estuarine surface waters

Abstract Two extraction procedures were examined, and it was found that DNA recovered from Campylobacter jejuni lysed by the cetyltrimethylammonium bromide (CTAB) method was more suitable for use as a PCR template than DNA released by the boiling method. The region targeted for PCR amplification was a 1.73-kb portion of the flagellin A gene of C. jejuni . The detection limit was lower than 30 cells per 100 ml in artificially contaminated waters. PCR assay and conventional culturing method had the same sensitivity, but results of the PCR technique were available within 48 h and so shortened the time necessary for detection by 48 h.     

Campylobacter jejuni: Specific oligonucleotides and DNA probes for use in polymerase chain reaction-based diagnosis

Abstract A 1189 base-pair long DNA fragment, VS1, was isolated from a Campylobacter jejuni CIP 70.2 cosmid library and was found to contain regions specific for this bacterial species. For detection and identification of C. jejuni , two oligonucleotides derived from the VS1 sequence were used as primers in polymerase chain reaction test on genomic DNAs from 38 Campylobacter and from 10 non- Campylobacter strains. A specific, 358 base-pair long DNA fragment was amplified only when C. jejuni DNA was used as a target. The detection limit of the amplification reaction was as low as 1.86 fg DNA, which is the equivalent of one C. jejuni genome.     

Application of conductance techniques to the detection of thermophilic Campylobacter spp. in river water

A study of basal media identified Campylobacter enrichment broth, with (CEB+) and without (CEB) antibiotic supplement, as a suitable medium for the detection and enumeration of Campylobacter jejuni, C. coli and C. lari within aqueous samples via conductance methodology. Despite apparent differences in conductivity profiles between species in the presence of antibiotics, no significant differences (P<0.05) were detected between detection times for each species tested. CEB+ was successfully employed within a combined enrichment and conductance protocol to the detection of C. jejuni from river water at a concentration of 1 CFU ml⁻¹ from 83% of samples in under 39 h and thus demonstrated an improvement over an applied conventional membrane filtration technique.     

安徽部分地区动物源弯曲菌的分离鉴定及多位点序列分型

【背景】弯曲菌是一种重要的食源性人兽共患病原菌,革兰氏阴性、微需氧、弯曲螺旋状。【目的】为了解安徽地区弯曲菌流行状况和分子遗传特征,对安徽6个不同地区动物源的弯曲菌进行分离鉴定,并研究分离株分子分型。【方法】通过形态学及培养特性观察、生化试验、PCR方法对菌株进行鉴定。以弯曲菌7个管家基因asp A、gln A、glt A、gly A、pgm、tkt和unc A为目的基因对分离株进行多位点序列分型,并制成遗传进化树。【结果】共分离到42株弯曲菌菌株,源自6个地区的分离株具有较为一致的形态特性和相似的生化特性。多位点序列分型结果显示,本研究中共获得32种ST型,共发现9种新的ST型(8190、8222、8223、8831、8833、8841、8832、8834和8843)和6个新的等位基因(gln A606、gln A607、glt A518、gly A680、pgm863和unc A541)。进化树结果显示,空肠弯曲菌与结肠弯曲菌遗传关系相差甚远,聚集归为两个大群,分别有5个分支和3个分支。【结论】安徽6个地区不同来源的空肠弯曲菌与结肠弯曲菌均有丰富的基因型,且没有明显优势的基因型。从遗传变异的角度来看,空肠弯曲菌复杂多样,结肠弯曲菌相对保守。     

不同DNA抽提方法对普通PCR和实时荧光定量PCR方法检测家蚕微孢子虫的影响

为了优选快速、 灵敏、 特异的家蚕微孢子虫Nosema bombycis分子检测方法和DNA抽提方法, 本文通过对家蚕微孢子虫TaqMan探针荧光定量PCR检测方法和SYBR Green荧光定量PCR检测方法的建立以及反应体系优化, 并与普通PCR方法进行比较; 再采用4种不同DNA抽提方法分别对PCR和实时荧光定量PCR方法检测家蚕微孢子虫悬浮液的效果评价。结果显示: 不经过DNA抽提, 直接将家蚕微孢子虫发芽液进行PCR反应的效果优于其他方法, 检测灵敏度由高到低依次为直接法、 酚/氯仿抽提法、 动物组织DNA试剂盒抽提法和植物组织DNA试剂盒抽提法; TaqMan探针法检测家蚕微孢子虫发芽液的灵敏度和SYBR Green法相近, 达到微孢子10²个/mL, 两者均优于普通PCR方法。实验表明, 直接采用发芽液结合荧光定量PCR方法检测家蚕微孢子虫最为简便、 快速、 灵敏。该研究结果将有助于提高家蚕微粒子病监控技术和检疫能力, 对家蚕微粒子病的检疫和防治具有积极意义。     

Quantification of Campylobacter spp. in chicken carcass rinse by real‐time PCR

Aims: In this study, a real‐time quantitative polymerase chain reaction (PCR) method was examined for its ability to quantify Campylobacter spp. in chicken carcass rinses and compared with bacteriological culturing. Methods and Results: The linearity of the real‐time PCR quantification protocol was assessed on pure DNA. The amplification efficiency was 100% and the square regression coefficient (R²) was 0·998. Quantification was linear over at least 7 log units. Using a crude cell lysate gave the highest sensitivity and the detection limit of the method was 3·3 log CFU per carcass. The statistical correlation between the bacteriological enumeration and the real‐time quantitative (Q)‐PCR determined using chicken carcasses sampled at the end of the slaughter line was 0·733. The difference in detection levels was probably because of the detection of stressed, dead or viable but not culturable cells by Q‐PCR. Conclusion: The real‐time Q‐PCR method described in this study is a powerful tool for determining the number of Campylobacter cells on carcasses. Significance and Impact of the Study: The real‐time Q‐PCR method is available to quantify the Campylobacter contamination at the slaughterhouse level and could be used to evaluate primary production.     

Quantitative detection of Helicobacter pylori in water samples by real-time PCR amplification of the cag pathogenicity island gene, cagE

Aims:  A new real-time PCR assay that simultaneously amplifies a 102-bp fragment of the cagE gene from Helicobacter pylori and a new internal positive control containing a specific sequence of the gyrB gene from Aeromonas hydrophila , was developed and validated for the detection of H. pylori in environmental samples.
Methods and Results:  The specificity, limits of detection and quantification, repeatability, reproducibility, and accuracy of the method were calculated. The resulting values confirmed the applicability of the method for the quantitative detection of H. pylori . The feasibility of the method was also evaluated by testing 13 pyloric antrum-positive biopsies and 69 water samples, including potable (10), surface (19) and wastewater (40) matrices. The results showed that all the biopsies and 3 of the 40 wastewater samples analysed were positive.
Conclusions:  This real-time PCR method provides a sensitive, specific, and accurate method for the rapid quantification of H. pylori in environmental samples.
Significance and Impact of the Study:  The PCR diagnostic system proposed in this work, provides a suitable tool for the quantitative detection of H. pylori in environmental samples and can be useful for verifying the role of water as a potential route of its transmission.     

Quantitative strain-specific detection of Lactobacillus rhamnosus GG in human faecal samples by real-time PCR

Aims:  To develop a strain-specific rapid assay for identification and quantification of Lactobacillus rhamnosus GG in human faecal samples.
Methods and Results:  A unique random amplified polymorphic DNA (RAPD) band of the L. rhamnosus GG strain was isolated and sequenced. Strain-specific polymerase chain reaction (PCR) primers and probes were designed based on the sequence. Quantification was performed by the real-time PCR using a fluorescent resonance energy transfer (FRET) system. The specificity of the assay was tested with DNA isolated from a set of known strains and human faecal samples. The analytical sensitivity of the method for L. rhamnosus GG was about 10 CFU per assay, which corresponds to 10⁵ CFU g⁻¹ of wet faeces.
Conclusions:  Quantitative real-time PCR is a suitable method for strain-specific identification of L. rhamnosus GG in human faecal samples.
Significance and Impact of the Study:  Lactobacillus rhamnosus GG is one of the most studied probiotic strains in clinical trials but still lacks a DNA-based identification method. This study describes a real-time PCR method for strain-specific identification and quantification of L. rhamnosus GG in human faecal samples.     

Molecular identification of Liriomyza trifolii (Burgess) (Dipt., Agromyzidae) based on real-time PCR

Abstract:  Rapidly identifying juvenile individuals of Liriomyza trifolii (Burgess) from Liriomyza sativae Blanchard is crucial in plant quarantine. We report a molecular method to identify L . trifolii based on real-time polymerase chain reaction (PCR). By comparing partial DNA sequences of mitochondrial COI genes of L . trifolii samples collected from Guangdong and Taiwan provinces in China, Japan, Philippine, Israel, Germany, the USA, Mexico and Honduras sequenced by authors, and those of related species recorded in GenBank, a L . trifolii -specific probe was developed. There was no difference in individuals of different stages tested by this probe. The total time for real-time PCR assay system was 2 h, and it would save 3–7 h compared with conventional PCR.     

Evaluation of PCR for detection of Campylobacter in a national broiler surveillance programme in Denmark

AIMS: To develop and evaluate a rapid and sensitive PCR method for detection of Campylobacter spp. directly from chicken faeces. METHODS AND RESULTS: DNA was isolated from faecal swabs using magnetic beads followed by PCR using a prealiquoted PCR mixture, which had been stored in the freezer. The result could be obtained in <6 h. The method was evaluated on 1282 samples from the Danish surveillance programme for Campylobacter in broilers by comparing with conventional culture. The diagnostic specificity was calculated to be 0.99. The detection limits of the PCR method and of the conventional culture were compared using spiked control material. For both methods the detection limit was 36 CFU ml-1. CONCLUSIONS: It was concluded that the PCR proved useful for detection of Campylobacter in pooled cloacal swabs from broilers. SIGNIFICANCE AND IMPACT OF THE STUDY: By taking cloacal samples in the broiler flocks the technique can be used as an important tool for planning and directing the broiler slaughtering process. This will be a great help in minimizing the risk of contaminating Campylobacter-free flocks at the abattoir.