Rapid and sensitive PCR detection of Vibrio penaeicida, the putative etiological agent of syndrome 93 in New Caledonia

Experimental infections of Penaeus (Litopenaeus) stylirostris were performed with a Vibrio penaeicida strain (AM101) isolated in New Caledonia from Syndrome 93 diseased shrimp. Cumulative mortalities resulting from intramuscular injection or immersion of shrimp in bacterial suspensions demonstrated high virulence for this bacterial strain and suggested that V. penaeicida could be the etiological agent of Syndrome 93. The median lethal dose (LD50) for AM101 was 1.3 x 10(4) CFU (colony forming units) ml-1 by immersion and less than 5 CFU shrimp-1 by intramuscular challenge, with mortality outbreaks at 48 and 22 h after challenge, respectively. A polymerase chain reaction (PCR) detection assay using a primer set designed from the 16S ribosomal RNA gene of V. penaeicida was developed. It gave an expected amplicon of approximately 310 bp in ethidium bromide-stained agarose gels. The specificity of these primers was assessed with different Vibrio species. Furthermore, DNA extracted by the Chelex method could be used to detect fewer than 20 cultured Vibrio cells in sea-water or shrimp hemolymph by this assay. It appears to be a reliable screening method for detecting V. penaeicida in shrimp and from the aquatic environment.     

Rapid identification of Bactrocera latifrons (Dipt., Tephritidae) by real-time PCR using SYBR Green chemistry

Abstract:  The solanum fruit fly, Bactrocera latifrons (Hendel), is a major agricultural pest in Asia and Hawaii, and it is important to prevent its widespread invasion in plant quarantine. In this study we introduced a real-time polymerase chain reaction (PCR) essay, using SYBR Green I dye, to rapidly identify B. latifrons on an ABI PRISM 7700 sequence detection system. A latifron-specific PCR primer set was obtained based on mtDNA COI gene of B. latifrons . Nine Bactrocera fruit flies, B. latifrons , Bactrocera dorsalis , Bactrocera papayae , Bactrocera carambolae , Bactrocera philippinensis , Bactrocera occipitalis , Bactrocera correcta , Bactrocera cucurbitae and Bactrocera tau , were used to determine the specificity of primers lati1 and lati2. A series of genomic DNA dilutions of B. latifrons (0.01, 0.1, 1, 10, 20, 40 and 100 ng) were used to assess the sensitivity of the SYBR Green PCR. Template DNA concentration was one of the sources of variability in cycle threshold values (CT) and the optimum DNA concentration was between 1 and 20 ng. Genomic DNA isolated from larvae, pupae and adult specimens of B. latifrons were used to assess the specificity of the SYBR Green PCR. Melting curve analysis and agarose gel electrophoresis was employed to check the specificity of PCR products. Similar amplification plots were obtained using DNA from the three different stages of B. latifrons with primer set lati1/lati2. The melting temperature ( T _m) of PCR products was 77.5 ± 0.1°C, and the length of the amplified fragment 366 bp. Given the specificity and sensitivity of the assay, combined with high speed, low cost and the possibility of automating, SYBR Green PCR can be used as a rapid and specific technique for pest species identification in plant quarantine.     

Development of a SYBR Green I real-time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood

AIM: Vibrio alginolyticus is an economically important micro-organism. The main aim of the present study was to develop a real-time polymerase chain reaction (PCR) assay for rapid, sensitive and effective quantification of V. alginolyticus in seawater and seafood. METHODS AND RESULTS: Purified DNA of V. alginolyticus, artificially inoculated seawater and seafood tissue homogenates were subjected to the gyrB-targeted real-time PCR assay. Natural seawater and seafood samples were analysed by this real-time PCR protocol. Specificity tests showed that positive result was obtained only with V. alginolyticus strains. The detection sensitivity was determined to be 0.4 pg of genomic DNA equivalent to 72 cells per PCR in pure culture and 100 cells in 1 ml of seawater or seafood tissue homogenates. Single cell detection is achieved after 3 h of sample enrichment. CONCLUSIONS: A sensitive and specific SYBR Green I-based real-time PCR assay targeting gyrB gene was successfully developed to quantify V. alginolyticus within 6 h in seawater and seafood samples. SIGNIFICANCE AND IMPACT OF THE STUDY: No report on the molecular-based method was available for quantitative detection of V. alginolyticus. This work will provide a novel method for evaluation of the risk of V. alginolyticus to marine environmental health and seafood safety.     

A real-time PCR assay for the detection and quantitation of Campylobacter jejuni using SYBR Green I and the LightCycler

Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional biochemical identification for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable (VNC) state. Nucleic acid-based tests have emerged as a useful alternative to traditional testing. In this article, we present fluorescent quantitative PCR assay for quantitative detection of C. jejuni, the assay was carried out using a LightCycler instrument and product formation was monitored continuously with the fluorescent double-stranded DNA binding dye SYBR Green I. When this assay was applied, the assay positive for all of the isolates of C. jejuni tested (11 isolates, including type strain ATCC33560) and negative for all other Campylobacter spp. (three isolates) and several other bacteria (five species tested). The total assay could be completed in 60 min with a detection limit of approximately 1 CFU, and a correlation coefficient was 1.000. Result indicated that fluorescent quantitative detection methods provided a special, sensitive, rapid, reproducible and accurate method for quantitative detection of C. jejuni.     

Quantitative detection of Proteus species by real-time polymerase chain reaction using SYBR Green I

A SYBR Green real-time polymerase chain reaction (PCR) method for rapid detection of Proteus species was developed and evaluated. Of 322 clinical and food samples tested, 75 samples were positive for Proteus species by using conventional PCR and real-time PCR assays. The results were consistent with standard culture methods and the Vitek auto-microbe system, indicating a 100 % specificity obtained by both PCR assays. For the real-time PCR method, the minimum detectable level was 10 colony forming units (CFU) /ml, which was a 10³ multiple higher than the conventional PCR method. Correlation coefficients of standard curves which were constructed using the threshold cycle (Ct) versus copy numbers of Proteus showed good linearity (R ²?=?0.997). In conclusion, several significant advantages such as higher sensitivity and rapidness were observed by using the SYBR Green real-time PCR method for identifying Proteus species.     

SYBR Green实时荧光定量PCR快速检测口腔幽门螺杆菌

目的建立一种快速、灵敏、特异的鉴定幽门螺杆菌实时荧光定量PCR方法。方法利用SYBR Green实时荧光定量PCR反应体系对口腔幽门螺杆菌进行检测。鉴定结果与临床常规鉴定方法相比较,评价其敏感度、特异度及重复性。结果通过48例样品的检测,结果显示实时荧光定量PCR法检测标本的鉴定结果与常规PCR鉴定方法的结果对比,特异度为100%,敏感度为100%;最小能检测到102个拷贝数的重组质粒;批内重复试验和批间重复试验结果均与常规鉴定方法结果相符。结论实时荧光定量PCR法鉴定口腔幽门螺杆菌,特异度和敏感度高,重复性好,且快速、简便。该方法有望成为检测口腔幽门螺杆菌感染的一种快速有效的方法。     

小麦根腐病菌索氏平脐蠕孢SYBR Green I实时荧光定量PCR检测技术研究

由索氏平脐蠕孢Bipolaris sorokiniana引起的小麦根腐病,常和其他土传真菌病害混合发生,传统的症状鉴别方法很难区分,导致病害防控难度增加。为建立病菌实时荧光定量检测体系,根据ITS序列设计引物,筛选出1对特异性引物BS‐F/R,扩增片段大小为280bp。以菌丝DNA为标准品构建实时荧光定量标准曲线,并对其灵敏度、特异性、可重复性进行评价。结果表明,建立的实时荧光定量PCR检测方法速度快,灵敏度高,特异性强,重复性好。构建的荧光定量PCR标准曲线循环阈值与模板浓度呈良好的线性关系,溶解曲线的吸收峰单一,扩增效率良好。利用该定量检测体系,可以检测出田间小麦样品中52.8fg/μL的病菌DNA。     

Tetraalkylammonium derivatives as real-time PCR enhancers and stabilizers of the qPCR mixtures containing SYBR Green I

Tetraalkylammonium (TAA) derivatives have been reported to serve as stabilizers of asymmetrical cyanine dyes in aqueous solutions and to increase the yield and efficiency of polymerase chain reaction (PCR) detected by end-point analysis. In this study, we compared the ability of various TAA derivatives (with alkyl chain ranging from 1 to 5 carbons) and some other compounds to serve as enhancers of real-time PCR based on fluorescence detection from intercalating dye SYBR Green I (SGI). Our data indicate that TAA chlorides and some other TAA derivatives serve as potent enhancers of SGI-monitored real-time PCR. Optimal results were obtained with 10-16 mM tetrapropylammonium chloride. The effect of TAA compounds was dependent on the nature of counter ions present and composition of the reaction mixtures used. Based on measurements of SGI-generated fluorescence signal in the presence of PCR-amplified DNA fragments, oligonucleotide primers and/or various additives, we propose that TAA-derivatives reduce the binding of SGI to oligonucleotide primers and thus enhance primer-template interactions during annealing phase. Furthermore, these compounds serve as stabilizers of SGI-containing PCR mixtures. The combined data indicate that TAA derivatives might be a new class of additives contributing to robustness of real-time PCR monitored by asymmetrical cyanine dye SGI.     

Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR

SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye and is included in many commercially available kits at undisclosed concentrations. Binding of SG to double-stranded DNA is non-specific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis because the melting temperature (T_m) of the specific amplicon is analogous to the detection of an electrophoretic band. When using SG for real-time PCR multiplex reactions, discrimination of amplicons should be possible, provided the T_m values are sufficiently different. Real-time multiplex assays for Vibrio cholerae and Legionella pneumophila using commercially available kits and in-house SG mastermixes have highlighted variability in performance characteristics, in particular the detection of only a single product as assessed by T_m analysis but multiple products as assessed by agarose gel electrophoresis. The detected T_m corresponds to the amplicon with the higher G+C% and larger size, suggesting preferential binding of SG during PCR and resulting in the failure to detect multiple amplicons in multiplex reactions when the amount of SG present is limiting. This has implications for the design and routine application of diagnostic real-time PCR assays employing SG.     

Quantification of Frankia in soils using SYBR Green based qPCR

A SYBR Green based qPCR method was developed for the quantification of clusters 1 and 3 of the actinomycete Frankia in soils. Primer nifHr158 was designed to be used as reverse primer in combination with forward primer nifHf1 specifically amplifying a 191-bp fragment of the nifH gene of these Frankia. The primer combination was tested for specificity on selected pure cultures, and by comparative sequence analyses of randomly selected clones of a clone library generated with these primers from soil DNA extracts. After adjustments of DNA extraction conditions, and the determination of extraction efficiencies used for sample normalization, copy numbers of nifH genes representing Frankia of clusters 1 and 3 were quantified in different mineral soils, resulting in cell density estimates for these Frankia of up to 10(6) cells [g soil {dry weight}](-1) depending on the soil. Despite indications that the nifH gene is not a perfect target for the quantification of Frankia, the qPCR method described here provides a new tool for the quantification and thus a more complete examination of the ecology of Frankia in soils.     

Rapid detection and differentiation of the major mycoplasma contaminants in cell cultures using real-time PCR with SYBR Green I and melting curve analysis

A quantitative real-time polymerase chain reaction (PCR) procedure followed by melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid detection and differentiation of mycoplasma contaminants in cell cultures. This method showed that the detection of the target sequence was linear over a range from 10(4) to 10 colony-forming units (CFU) of the mycoplasma cells. Analysis of the melting temperature of the PCR products allowed differentiation of the major mycoplasma contaminants. These results demonstrate that the protocol described in the present study can decrease the time to obtain reproducible results by simultaneous detection and differentiation of the Mycoplasma species contaminating cell cultures.     

Rapid zygosity determination in mice by SYBR Green real-time genomic PCR of a crude DNA solution

We examined whether crude DNA extracts prepared from gene-engineered mouse tissues are suitable as a template for zygosity determination by SYBR Green real-time genomic PCR. A crude DNA solution was prepared by brief incubation with lysis buffer containing ear, tail, or fetus of ROSA26 mouse, a gene-trapped strain carrying the β-galactosidase (β-gal) gene. Five serially diluted crude DNA samples (original, 2-, 4-, 8-, 16-diluted) were next prepared and then subjected to three-step (95°C, 60°C and 72°C) reactions of real-time PCR to detect the β-gal gene and the receptor-activity-modifying protein 3 (ramp3) gene (as an internal reference gene). The slopes of standard curves obtained from the real-time PCR indicated that amplification efficiency was approximately 99%, and the efficiencies of target and reference were almost equal. With this system, we next determined the zygosity of mice derived from mating heterozygous ROSA26 females and males, and found a sharp distinction in zygosity, wild-type, heterozygous and homozygous. Assessment of crude DNA samples from other gene-engineered mice including B6ZP3Cre-Tg, B6rAM-Tg, and Ramp2-gene-targeted strains revealed that our method was effective for determination of zygosity. The present method is more convenient and rapid than formerly published methods employing purified genomic DNA as a template. Our method will be particularly useful for experiments requiring rapid and accurate genotyping of gene-modified animals/fetuses.     

Evaluation of touch-down real-time PCR based on SYBR Green I fluorescent dye for the detection of Neisseria meningitidis in clinical samples

Antibiotic treatment prior to transport or admission of patients to hospital has reduced the proportion of patients with invasive meningococcal disease (IMD) from whom Neisseria meningitidis can be isolated by standard microbiological techniques. Assays to detect the crgA gene were used to detect meningococcal DNA by both conventional polymerase chain reaction (PCR) and real-time PCR (RTPCR) in relation to microbiological diagnosis of cases over two years between 2002 and 2003. The sensitivity of both PCR assays for culture-confirmed cases was 93% and the specificity was 98.6%. Agreement between the two PCR assays was 96.2%. The inter- and intra-assay variations and effects of different amounts of DNA on the melting temperatures were examined. The touch-down RTPCR based on SYBR Green I fluorescent dye detected and characterized N. meningitidis in clinical samples within one hour.     

SYBR Green I荧光RT-PCR法检测贝类中的诺如病毒

针对诺如病毒II型的保守区域设计引物,建立了SYBR Green I实时荧光RT-PCR检测诺如病毒II型的反应体系。此方法的病毒检测下限达到102拷贝,标准曲线的线形范围为102~106拷贝,相关系数为0.9952,斜率为?2.982,截距为35.84。对诺如病毒II型检测特异,与轮状病毒、腺病毒、甲肝病毒、星状病毒无交叉反应。针对质粒标准品检测的批内试验变异系数 (CV) 为0.95％~1.69％ (n=5),批间试验CV为0.87％~1.24％ (n=3)。运用此方法随机检测30份贝类水产品,检测出2份阳性样品。结果表明,SYBR Green I荧光RT-PCR检测诺如病毒II型的方法灵敏、特异、重复性好,可应用于贝类水产品的快速检测。     

A quantitative real-time PCR assay for the detection of tetR of Tn10 in Escherichia coli using SYBR Green and the Opticon

Bacteria of implant infections are extremely resistant to antibiotics. One reason for this antibiotic resistance are transposons; the well-known transposon Tn10, for example, mediates tetracycline resistance to Escherichia coli. Two genes of Tn10, tetA and tetR, are essential for the mechanism of resistance. These genes encode a drug-specific efflux protein and a tetracycline repressor protein, respectively. Tn10 is also widely used in molecular biology. For example, tTA, a recombinant derivate of tetR, has been utilised for a highly efficient gene regulation system in mammalian cells. We have examined E. coli isolates from implant infections for tetracycline resistance and for the presence of tetR. A real-time PCR assay was developed for detection of tetR with SybrGreen using the Opticon PCR machine of MJ Research. This method offers a quick, sensitive, efficient, and reliable approach to the detection and quantification of genes. Clinical isolates of E. coli were examined successfully for tetracycline resistance and for the presence of tetR. The real-time PCR is effective using a variety of templates including isolated E. coli DNA, pure colonies, or liquid culture sources. Using quantified standard DNA, this assay can accurately detect as few as 15 copies. Moreover, this assay has the ability to quantify the number of tetR genes in the presence of contaminating mammalian DNA. In conclusion, the tetR real-time PCR offers new methods for detection and quantification of tetracycline-resistant bacteria and tTA in transfected cell-lines or transgenic animals.     

Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria

Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.