Simultaneous PCR amplification of two barley 5S rDNA sequence types in the same PCR reaction

Many researchers are currently using PCR technology to amplify individual members of multigene families, including 5S rDNA, for sequencing and related purposes. When members of the family differ in length, analyses would be facilitated if different units could be simultaneously and efficiently amplified. In the present paper we describe conditions that can be used to amplify simultaneously both the “long” and “short” 5S rDNA repeats found in barley (Hordeum vulgare L.).     

非幽门螺杆菌：一类人兽共患病病原菌

过去的几十年，非幽门螺旋杆菌的研究迅速发展，他们不仅能感染和人类密切相关的动物，在人类的胃肠、肝胆及其他系统的疾病中也起着重要的作用，是一类人兽共患病病原菌。     

Fluorescent Pseudomonas species categorized by using polymerase chain reaction (PCR)/restriction fragment analysis of 16S rDNA

A rapid procedure for the identification of fluorescent pseudomonads, based on the polymerase chain reaction (PCR) and restriction fragment analysis of 16S rDNA genes is described. Thirty-one strains belonging to 10 different Pseudomonas species of the Pseudomonas fluorescens rRNA branch were characterized. Amplified rDNA was digested with 13 different restriction endonucleases. The combined data from restriction analysis enabled the definition of 17 different 16S rDNA genotypes. All type strains belonging to different species were differentiated. The good correlation between grouping obtained using restriction analysis with other molecular classification criteria demonstrates the value of the described method to characterize rapidly fluorescent Pseudomonas strains at the species level.     

Development of a PCR-based technique for detection of Helicobacter pylori

Abstract A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori , using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.     

普通Taq DNA聚合酶扩增幽门螺杆菌尿素酶基因长片段

ＰＣＲ反应扩增较长的ＤＮＡ片段比较困难。本实验对幽门螺杆菌染色体ＤＮＡ上的２７Ｋｂ尿素酶基因用普通ＴａｑＤＮＡ聚合酶进行扩增,扩增结果以琼脂糖凝胶电泳证实,得到所需的２７Ｋｂ的片段。该实验为今后幽门螺杆菌工作的进一步研究提供了经济,有效,简捷的条件。     

口腔中幽门螺杆菌PCR检测方法的建立

全世界大约有50%的人口感染幽门螺杆菌(Helicobacter pylori, Hp),Hp感染容易造成慢性胃炎、消化性溃疡、低度恶性胃MALT淋巴瘤及胃癌,对人类造成严重的危害。为寻求一种简便、准确、非创伤性的方法用于口腔中Hp的检测,本研究建立了口腔中幽门螺杆菌的PCR检测方法,并对建立的PCR方法的敏感性、特异性进行分析,并应用于检测唾液样品和牙菌斑共50份。结果显示,50份样品中,阳性率为82%。结果表明,本研究建立的口腔中Hp的PCR检测方法特异性强、敏感性高,可以用于临床上Hp诊断的重要参考。     

Detection of Various Variant Verotoxin Genes in Escherichia coli by Polymerase Chain Reaction

We constructed common primers for the polymerase chain reaction to detect the genes for various Verotoxins reported, that is, VT1 (or SLT-I), VT2 (or SLT-II), VT2vha, VT2vhb, SLT-IIv (or VT2vp1, VTe) and SLT-IIva (or VT2vp2). A total of 80 Verocytotoxin-producing Escherichia coli strains isolated from humans, domestic animals and meats gave a positive result by PCR with the designed common primers. Digestion by restriction endonucleases BglII and EcoT14I of the amplicon of the VT2vp2 gene gave specific bands of the expected sizes, but not of the amplicons of other VT genes, suggesting a possible method for identification of the VT2vp2 gene. Application of the PCR with the designed primers in diagnostic and epidemiological studies on VTEC infection is also discussed.     

Application of real-time PCR stool assay for Helicobacter pylori detection and clarithromycin susceptibility testing in Brazilian children

Background: Helicobacter pylori ClariRes assay is a novel commercially available real‐time PCR assay allowing H. pylori detection and clarithromycin susceptibility testing in either gastric biopsy or stool specimens. Objective: The aim of this study was to validate the novel biprobe real‐time assay in stool specimens from 217 dyspeptic children. Methods: DNA from gastric biopsies and stool specimens were obtained and submitted to the biprobe real time assay for H. pylori detection and clarithromycin susceptibility testing. Results: The sensitivity, specificity, and test accuracy were 69, 100 and 93.9% for the detection of H. pylori infection and 83.3, 100 and 95.6%, for detection of clarithromycin resistance. Conclusion: This assay proved to be appropriate for H. pylori clarithromycin susceptibility testing, particularly in children populations where a high prevalence of clarithromycin‐resistant strains is suspected.     

Detection of Helicobacter pylori in water by direct PCR

AIMS: This paper demonstrates a rapid, simple method for the detection of Helicobacter pylori in water that eliminates the need for recovery of cells or DNA extraction prior to PCR. METHODS AND RESULTS: Direct polymerase chain reaction (DPCR) with primers specific for H. pylori ureA (urease, subunit A) were used to detect H. pylori added to groundwater. DPCR also detected H. pylori in a naturally contaminated water sample. CONCLUSIONS: DPCR should provide an improved method to assess contamination of water by H. pylori. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple, rapid method for detection of H. pylori in water will provide an improved means to investigate the possible role of water as a disease vector.     

A simple multiplex PCR assay for diagnosing virulent Helicobacter pylori infection in human gastric biopsy specimens from subjects with gastric carcinoma and other gastro-duodenal diseases

AIM: To evaluate and develop a multiplex polymerase chain reaction (PCR) assay for diagnosing and specific identification of virulent Helicobacter pylori strains and their main virulence genes cagA, cagE, cagT, vacA and hrgA. METHODS AND RESULTS: Genomic DNA from 82 gastric tissues was screened. A master pool of all the ingredients of multiplex reaction was prepared for amplification. Amplicons were sequenced to confirm the amplification of each target genes. Multiplex PCR assay was able to detect all the five target genes in 81.7% and deletions in one or more loci among 18.3%. Genotype cagT +ve/hrgA +ve/cagA +ve/cagE +ve/vacAs1 +ve was more predominant in this study population (67.07%). hrgA, cagT, cagE and cagA genes were present in 100%, 92.7%, 85.4% and 81.7% of the subjects, respectively. The vacAs1 subtype had higher prevalence frequency in patients with overt gastrointestinal disease (78.57%) than with GERD (gastro-esophageal reflux disease) and NUD (non-ulcer dispepsia) (50%). CONCLUSIONS: The multiplex PCR assay developed herein was able to genotype H. pylori isolates based on the main virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify H. pylori and the majority of their virulence gene markers by multiplex PCR assay represents a considerable advancement over other PCR-based methods for genotyping H. pylori from large population, and can be explored to gain insights at the genotypic variability exhibited by this pathogen.     

16S rDNA Sequence Analysis of Bacterial Isolates from Die-back Affected Sissoo Trees (Dalbergia sissoo Roxb.) in Bangladesh

A new form of disease called ‘die‐back’ has been established in Dalbergia sissoo trees. This disease has reached epidemic proportions in Bangladesh as well as in other countries of South Asia and is characterized by browning of the leaves, signs of wilting, and trunk lesions with gum flow. The trees die within a few months. In order to investigate the causes of this die‐back disease, samples were taken for a first trial in the Rajshahi division at two sites around Sherpur. For the isolation of bacteria, surface‐sterilized plant material (leaves, twigs and trunk bark) from diseased trees was transferred to LB medium and incubated. After isolation of single colonies, various bacteria species could be identified by polymerase chain reaction analysis with two primers specific for highly conserved sequence regions in the bacterial 16S rDNA and by sequencing. First indications for the presence of bacteria with phytopathogenic potential were found.     

Detection of Helicobacter pylori DNA by a simple stool PCR method in adult dyspeptic patients

INTRODUCTION: Helicobacter pylori is the major agent causing peptic ulcer, gastric cancer and mucosa-associated lymphoid tissue (MALT) gastric lymphoma. A simple stool polymerase chain reaction (PCR) method was performed and compared with the gold standards for the diagnosis of H. pylori infection. MATERIAL AND METHODS: A total of 54 adult patients (mean age, 46.41 +/- 13.12 years) with dyspeptic symptoms from Gastroenterology at Dokuz Eylül University Hospital between May and November 2003 were included. Two antrum and corpus biopsies were taken from each patient. Infection by H. pylori was defined as positivity and negativity of the gold standards. DNA extraction of stool specimens was done using QIAamp DNA Stool Mini Kit (QIAGEN) and PCR conditions included amplification and reamplification steps using the H. pylori ureA gene specific primers (HPU1, HPU2) and were visualized on 1% agarose gel stained with ethidium bromide. RESULTS: Forty-six of 54 patients (85.2%) were diagnosed positive and eight (14.8%) were negative for H. pylori infection by the gold standard methods. Thirty-two patients were positive (59.3%) and 22 of them (40.7%) were detected negative by stool PCR method. The stool PCR method and gold standard methods showed a statistical difference for the detection of H. pylori infection (p < .0001). Sensitivity, specificity, likelihood ratio, and positive and negative predictive values were 65.22%, 75%, 2.61%, 93.75%, and 27.7%, respectively. DISCUSSION: The PCR on the stool specimens resulted as being a very specific test. We suggest that a simple stool PCR method that we developed can be used to detect H. pylori, virulence genes, and in drug resistance studies either first line diagnostic methods in the laboratory or in the clinical management of dyspeptic patients.     

Detection of Mycobacterium avium ssp. paratuberculosis in cheese, milk powder and milk using IS900 and f57-based qPCR assays

Aims: To develop a quantitative PCR assay for sensitive and specific detection of Mycobacterium avium ssp. paratuberculosis (Map) in a range of dairy products. Methods and Results: TaqMan^® assays were designed to target the IS900 and f57 genetic elements of Map. Both real‐time PCR assays were integrated with the Adiapure^® Map DNA extraction kit and assessed separately for the detection/quantification of Map in spiked milk, Cheddar cheese and milk powder. Assays were validated against Cheddar cheese samples containing known concentrations of Map. The IS900 qPCR assay was significantly more sensitive than the assay based on the f57 primer/probe. At a threshold cycle value of 38, limits of detection (LOD) for the IS900 qPCR assay were 0·6 CFU ml^?1, 2·8 CFU g^?1 and 30 CFU g^?1 for artificially contaminated pasteurized milk, whole milk powder and Cheddar cheese, respectively. The respective LOD’s for the f57 assay were 6·2 CFU ml^?1, 26·7 CFU g^?1 and 316 CFU g^?1. Conclusion: The integrated Adiapure^® extraction – IS900 real time assay described is a sensitive, quantitative method for the detection of Map in dairy products. This is the first study to consider qPCR as a quantitative estimation of Map‐DNA in cheese and whole milk powder. Significance and Impact of the Study: The assay developed allows sensitive detection and quantification of Map DNA in a range of dairy products which is valuable for the screening and surveillance of this potential zoonotic organism.     

The Sequence of the Hartmannella vermiformis Small Subunit rRNA Coding Region

ABSTRACT The Hartmannella vermiformis small-subunit rRNA coding region was amplified, and the amplified DNA was cloned and sequenced. The coding region is 1,840 nucleotides long, and is typical of eukaryotic rRNA genes in both size and composition. Different clones contained different nucleotides at three positions.     

应用PCR技术检测病人血液标本中斑点热群立克次体DNA

本文以聚合酶链反应（PCR）,采用二次扩增法直接检测蜱传斑点热病人血液标本中斑点热群立克次体DNA,结果从7份标本中检出阳性1份,进一步的限制性片段多态性分析证明和斑点热群黑龙江立克次体基因型相同。实验证明：黑龙江立克次体对人具有致病性,同时也证明PCR技术快速、简单、敏感,用于斑点热群立克次体的早期诊断是可行的,并可作出分型鉴定。     

Helicobacter pylori stimulates a mixed adaptive immune response with a strong T-regulatory component in human gastric mucosa

BACKGROUND: Host factors play an important role in the pathophysiology of Helicobacter pylori infection and development of gastritis and related disease. The established opinion is that the T-cell-mediated immune response to H. pylori infection is of Th1 type. Our earlier immune cell phenotype studies indicate a mixed Th1-Th2 profile of the effector cells. Therefore, an extensive adaptive and regulatory cytokine gene expression profile was conducted by quantitative real-time polymerase chain reaction (qPCR). MATERIALS AND METHODS: Biopsies from gastric mucosa of 91 patients diagnosed as H. pylori negative, H. pylori positive with gastritis, or H. pylori positive with peptic ulcer were obtained by endoscopy. Gene expressions of nine cytokines and CagA status were measured by qPCR. RESULTS: All cytokine genes showed higher expression levels in the presence of H. pylori when compared to H. pylori-negative samples (fold increase: IL8: x 11.2; IL12A: x 2.4; TNF-alpha: x 5.2; IFN-gamma: x 4.3; IL4: x 3.6; IL6: x 14.7; and IL10: x 6.7). Patients infected with CagA-positive strains had higher expression of IL1-beta and IL18 compared to patients infected with CagA-negative strains (x 1.6 for IL1-beta and x 2.0 for IL18). Patients with duodenal ulcer had a lower antral Th1/Th2 ratio than other H. pylori-positive patients. CONCLUSIONS: The cytokine profile of H. pylori-infected gastric mucosa shows a mixed Th1-Th2 profile. Furthermore, a high IL10 expression may indicate that also regulatory T cells play a role in the chronic phase of H. pylori infection.     

Helicobacter pylori in Japanese river water and its prevalence in Japanese children

AIMS: The major transmission route of Helicobacter pylori remains unclear. In this study, we examined H. pylori in the environmental waters in Japan. METHODS AND RESULTS: A total of 24 water samples were collected from the upper, middle and downstream reaches of four Japanese rivers. Helicobacter pylori-specific DNA was examined using nested PCR. In addition, 224 children who lived near one river were studied by the stool antigen test for H. pylori prevalence. Helicobacter pylori DNA was detected in the water from the middle and downstream reaches of all four rivers, but not in the upper reaches. Helicobacter pylori was not found in cultured water samples with positive PCR results. Helicobacter pylori prevalence in the children examined was 9.8% for those living near the middle reaches and 23.8% nearby downstream, both of which were higher than the value in an area distant from the river (0%) (both, P < 0.01). CONCLUSIONS: Difference in H. pylori prevalence in the children may be related to the presence of H. pylori in the river. The results of this study showed that H. pylori DNA is frequently present in river water from the middle and downstream reaches in which the human biosphere is embedded. SIGNIFICANCE AND IMPACT OF THE STUDY: It is suggested that river water in the natural environment could be a risk factor for H. pylori transmission.     

Detection of Helicobacter pylori DNA in feces and saliva by polymerase chain reaction: a review

The polymerase chain reaction (PCR), known for its high sensitivity and specificity, has been used for the detection of Helicobacter pylori DNA in bodily materials such as feces and saliva. Since fecal specimens contain PCR inhibitors, DNA before PCR amplification has been purified using various biochemical, immunological and physical pre-PCR steps. Several PCR protocols, differing from each other in the selection of genomic targets and primers, have produced varying degrees of specificity and sensitivity in detecting H. pylori DNA. PCR identified antimicrobial resistance of H. pylori in feces. It also detected virulence factor genes such as the cytotoxin-associated gene (cagA) and vacuolating cytotoxin gene (vacA) in feces and saliva. While the cagA gene was detected in 50-60% of fecal specimens, it was found in 25% of salivary specimens from patients. There was considerable variation in the detection rate of H. pylori DNA in salivary samples. The detection rate in saliva with the most effective primer pair was lower than that observed in feces, making saliva a less suitable specimen for the diagnosis of H. pylori infection. There is controversy regarding the permanent presence of H. pylori in saliva. Whether the salivary and gastric specimens of an individual harbor identical or different strains has not been resolved. PCR cannot distinguish between living and dead organisms. However, it can offer quick results on fecal and salivary specimens, which may contain fastidious and slow-growing H. pylori in low numbers.