禽大肠杆菌、肠炎、鼠伤寒、鸡白痢及鸡伤寒沙门菌多重PCR方法的建立及应用

【背景】大肠杆菌病和沙门菌病是最常见的家禽细菌性疾病,给养禽业造成严重经济损失。另外,禽大肠杆菌和沙门菌也是重要的人畜共患病原菌,可通过禽类及其产品传播给人类,对人类健康造成严重威胁。加强禽大肠杆菌和沙门菌的快速鉴别检测,对养禽业和公共卫生都具有重要意义。【目的】建立禽大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的多重PCR检测方法。【方法】通过比较分析确定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的特异靶标基因,设计5对特异性引物,通过条件优化建立多重PCR方法,分析该多重PCR方法的特异性、敏感性及可靠性。【结果】该方法能特异性地鉴定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌,每个PCR反应的最低检出限分别为103 CFU细菌和100 pg基因组DNA。临床分离菌株检测显示,多重PCR与传统血清学方法结果一致。【结论】建立的多重PCR方法能够快速鉴别禽致病性大肠杆菌和不同血清型沙门菌,对禽大肠杆菌病和沙门菌病的流行病学调查及临床检测具有重要意义。     

Differentiation of Salmonella enterica serotype gallinarum biotype pullorum from biotype gallinarum by analysis of phase 1 flagellin C gene (fliC)

Salmonella enterica serotype gallinarum biotype gallinarum and biotype pullorum are non-motile and pathogenic avian strains. Biotype gallinarum causes fowl typhoid and biotype pullorum is the cause of pullorum disease in chickens. The two biotypes could be differentiated based on biochemical characteristics. However, conventional culture and biochemical assays are time-consuming, laborious and need sterile laboratory practices. Although the two biotypes, gallinarum and pullorum are non-motile, they possess the phase 1 flagellin C gene. The variable regions of the flagellin C gene from 41 biotype pullorum and 52 biotype gallinarum were amplified by colony-PCR and analyzed by single strand conformational polymorphism (SSCP) method. Differences in SSCP electrophoretic patterns were confirmed by nucleotide sequencing. In addition, PCR-RFLP with Hinp1I was also successfully applied to differentiate the two biotypes. These results suggested that the variable regions of fliC could be used as a genetic marker to differentiate biotype gallinarum from biotype pullorum.     

Determination method of sense sequence based on RT/PCR

When novel sequences are isolated by differential display and other methods, it seems useful to determine which is a sense sequence at an early stage before further experiments. A novel sequence, named MT-001, which shows enhanced expression in the permanent ischemic rat brain, was isolated by differential display. Based on this sequence, a primer set for both direction was designed. Each primer was used to make a cDNA and PCRs performed with each cDNA and both primers. One primer used in the RT step produced a PCR product at the expected position, but another primer in the reverse direction could not. This result indicated that the primer that made the expected PCR product is antisense.     

PCR method based on the ogdH gene for the detection of Salmonella spp. from chicken meat samples

In a previous paper, the ogdH gene that encodes 2-oxoglutarate dehydrogenase was isolated from Salmonella typhimurium. The catalytic N-terminal region in the enzyme was found to be very specific for the Salmonella species. Therefore, the aim of the present study was to detect S. typhimurium in food sources using primers designed for OGDH-1 and OGDH-2 which were based on the salmonella-specific region of the ogdH gene. A simple polymerase chain reaction (PCR) detection method was developed to detect low numbers of S. typhimurium in a chicken meat microbial consortium. Using the ogdH-specific primers under stringent amplification conditions and for gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the PCR assay was run on S. typhimurium-contaminated meat contents, only the positive meat samples containing as few as 200 CFUs reacted to the assay. The method employed for sample processing is simple and it was determined to provide a sensitive means of detecting trace amounts of S. typhimurium-specific sequences in the presence of mixed meat microbial populations. When compared with six representative intestinal gram-negative bacterial strains in foods, including Vibrio parahaemolyticus, V. vulnificus, Enterobacter cloacae, E. coli O157:H7, Pseudomonas aeruginosa, and Proteus sp., S. typhimurium had a unique and distinct PCR product (796 bp). In conclusion, the two OGDH primers were found to be rapid and sensitive detectors of Salmonella spp for the PCR method.     

PCR amplification of the fimA gene sequence of Salmonella typhimurium, a specific method for detection of Salmonella spp.

The goal of this study was to evaluate the suitability of the fimA gene amplification by PCR as a specific method for detection of Salmonella strains. Salmonella typhimurium and other pathogenic members of the family Enterobacteriaceae produce morphologically and antigenically related, thin, aggregative, type 1 fimbriae. A single gene, fimA, encodes the major fimbrial unit. In order to obtain higher specificity, we have selected a series of primers internal to the fimA gene sequence and have developed a PCR method for detecting Salmonella strains. A collection of 376 strains of Salmonella comprising over 80 serovars, isolated from animals and humans in Canada, have been used to evaluate this PCR method. Forty non-Salmonella strains were also tested by the same procedure. Cultures were screened by inoculating a single colony of bacteria directly into a PCR mixture containing a pair of primers specific for the fimA gene. The specific PCR product is an 85-bp fragment which was visualized by polyacrylamide gel electrophoresis and ethidium bromide staining. All Salmonella strains gave positive results by the PCR. Feed and milk samples contaminated by Salmonella strains were also detected by this procedure. The detection of all Salmonella strains tested and the failure to amplify the fragment from non-Salmonella strains confirm that the fimA gene contains sequences unique to Salmonella strains and demonstrate that this gene is a suitable PCR target for detection of Salmonella strains in food samples.     

Allele-specific long-range PCR/sequencing method for allelic assignment of multiple single nucleotide polymorphisms

We report an allele-specific sequencing method using allele-specific long-range polymerase chain reaction (PCR) to determine if multiple (specifically, more than three) single nucleotide polymorphisms (SNPs) are located on the same allele. We sequenced the glucocorticoid receptor (GR) gene as a model and detected four nucleotide changes, including two novel variations, in intron 4 and exons 6, 8, and 9 alpha in four of the investigated cell lines. The terminal SNPs (intron 4 and exon 9 alpha) were separated by 19 kb. Following SNP identification, the first round PCR allele-specific primers are designed at the both distal SNP sites (intron 4 and exon 9 alpha), placing the SNP positions at the primer 3'-end. Using these first round PCR products as template, the second round PCR was performed to separately amplify exons 6 and 8. These second round PCR products were subsequently sequenced. The sequencing results showed that the four SNPs were located on the same allele, i.e., forming a haplotype. This allele-specific long-range PCR/sequencing (ALP/S) method is rapid and applicable to the allelic assignment for more than three SNPs.     

An Allele-specific PCR Assay for Detecting Azoxystrobin-resistant Alternaria Isolates from Pistachio in California

Alternaria late blight caused by Alternaria spp. in the alternata, tenuissima and arborescens species‐groups is one of the most common fungal diseases of pistachio in California. A single point mutation resulting in the replacement of a glycine by an alanine at codon 143 (G143A) in the mitochondrial cytochrome b (cyt b) has been found in all azoxystrobin‐resistant isolates of these three species from California pistachio. In this study, a pair of allele‐specific polymerase chain reaction (PCR) primers was developed to detect this point mutation. The allele‐specific PCR assay coupled with a rapid DNA extraction method could detect azoxystrobin‐resistant Alternaria isolates in a few hours. The allele‐specific PCR method was also reliable for detecting azoxystrobin‐resistant Alternaria directly in both laboratory‐inoculated and naturally infected pistachio leaves.     

Extracellular superoxide dismutase polymorphism in mice: Allele-specific effects on phenotype

Extracellular superoxide dismutase (ecSOD) protects the extracellular matrix from oxidative stress. We previously reported a new allele for ecSOD, expressed in 129P3/J mice (129), which differs from the wild type (wt), expressed in C57BL/6J and other strains, by two amino acid substitutions and a 10-bp deletion in the 3′ UTR of the mRNA (A. Pierce et al., 2003, Arterioscler. Thromb. Vasc. Biol. 23:1820–1825). The newly discovered allele is associated with a phenotype of significantly increased circulating and heparin-releasable enzyme activities and levels. To examine the properties of the two forms of ecSOD in an identical environment we generated, by extensive backcrossing of ecSOD heterozygous progeny to C57BL/6J females, a congenic C57 strain with the 129 (or wt) allele of ecSOD. These mice are homozygous for nearly 5000 SNPs across all chromosomes, as determined by the Affymetrix Parallele Mouse 5K SNP panel. This study describes the generation of the congenic mice (genetically > 99.8% identical) and their ecSOD phenotype. The congenic mouse plasma ecSOD activity before and after heparin administration recapitulates the differences reported in the founder mice. Tissue enzyme distribution is similar in both congenic groups, although the 129 allele is associated with higher levels of enzyme expression despite lower levels of enzyme mRNA. In these characteristics the phenotype is allele driven, with little impact from the rest of the genome. The congenic mice carrying the 129 allele have mRNA levels that are in between those in the founder 129P3/J and C57BL/6J strains. We conclude that the ecSOD phenotype in most aspects of enzyme expression is allele driven, with the exception of tissue mRNA levels, for which a significant contribution by the surrounding (host) genome is observed. These results also suggest potential allele-specific differences in the regulation of ecSOD synthesis and intracellular processing/secretion of ecSOD, independent of the genotype context. Most importantly, the congenic mice offer an excellent model to examine the regulatory mechanisms of ecSOD expression and the role of ecSOD in various diseases involving oxidative stress.     

Allele-specific polymerase chain reaction: a method for amplification and sequence determination of a single component among a mixture of sequence variants

A new technique is described for amplifying individual alleles in a mixture of two or more alleles by the polymerase chain reaction (PCR) to determine their nucleotide sequence. This technique involves amplifying and separating target sequences by the PCR-mediated single-strand conformation polymorphism (PCR-SSCP) method, isolating each polymorphic DNA strand, and amplifying it by a second-stage PCR for its sequence determination. By this technique, the sequence of a minor constituent (approximately 3%) can be determined accurately.     

A genomic walking method for screening sequence length polymorphism

We adapted a recently developed nonrestrictional, nonligational genome walking method, Universal Fast Walking (UFW), for detection of length polymorphism in the proximal promoter region of genes. We demonstrate its efficacy at discovering naturally occurring transposition into heat‐shock genes of wild Drosophila and show that it surmounts limitations of simple polymerase chain reaction (PCR) approaches. We further present modifications to the standard UFW protocol and provide some guidelines to improve specificity. Although the resultant banding pattern of a standard UFW can be regarded as a DNA fingerprint, many amplicons result from false priming and not real polymorphisms. We describe ways to distinguish between UFW amplicons and false priming products in a high‐throughput assay.     

Real-time PCR method for Salmonella spp. targeting the stn gene

AIM: To develop a real-time PCR assay for Salmonella spp. targeting the stn gene. METHODS AND RESULTS: The presence of stn in the Salmonella bongori genome was found by a BLAST with Salmonella enterica stn sequence. Manual alignment of stn sequences showed that Salm. bongori had 88% sequence identity with Salm. enterica. Two primers (stnL-433 and stnR-561) and a probe (stnP-452) were designed to target conserved regions in stn and meet the requirements of a 5'-nuclease assay. The primers and probe were evaluated against 353 isolates, including 255 Salm. enterica representing 158 serotypes, 14 Salm. bongori representing 12 serotypes and 84 non-Salmonella representing 56 species from 31 genera. All isolates were correctly identified, with the exception of three isolates of Citrobacter amalonaticus, which gave false positives. The limit of detection with cultured Salmonella was 3 CFU per reaction. CONCLUSIONS: The stn real-time PCR method had 100% inclusivity, 96.4% exclusivity and a level of detection of 3 CFU per reaction for cultured Salmonella spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that stn is present in Salm. bongori and is a valid target for both species of Salmonella. The Salmonella s tn real-time PCR is a useful method for identifying Salmonella spp.     

A rapid method for purifying PCR products for direct sequence analysis

An HPLC approach for purification and sequencing of double-stranded DNA obtained directly from a PCR is described. This simple and reliable procedure has several advantages; the DNA fragment is rapidly eluted (less than 7 minutes), requires no organic cleanup, produces several hundred bases of sequence and is sensitive enough to obtain DNA sequence from a single 100-microliters PCR. This method is demonstrated by sequencing tumor necrosis factor alpha (TNF alpha) gene amplified from mouse tail DNA.     

Cloning and DNA sequence analysis of the serC-aroA operon from Salmonella gallinarum; evolutionary relationships between the prokaryotic and eukaryotic aroA-encoded enzymes

The serC-aroA operon of Salmonella gallinarum was isolated from a gene library using a labelled oligonucleotide probe and by complementation of an aroA Escherichia coli strain. The nucleotide sequence of a 2.6 kbp fragment was determined. The predicted amino acid sequence of the aroA gene product was compared to the equivalent sequence from ten other organisms. Computer-generated evolutionary trees clearly divide the eleven sequences into four different groups: Gram-negative bacteria, Gram-positive bacteria, fungi and plants. These trees depict a close evolutionary relationship between the sequences from Gram-negative bacteria and higher plants.     

Methods for detecting single nucleotide polymorphisms: Allele-specific PCR and hybridization with oligonucleotide probe

The existing diversity of the methods for detecting single nucleotide polymorphisms is so great that may perplex an unsophisticated researcher who chooses the appropriate molecular genetic toolkit. In this work, we tried to systematize and briefly describe the state-of-the-art methods for detecting oligonucleotide polymorphisms that are based on allele-specific PCR and hybridization with oligonucleotide probe as well as to characterize the methods considered with respect to their accuracy, cost, and simplicity.     

Biologically-generated primer for PCR: PCR primer of unknown sequence.

We describe a method for producing specific PCR primers directly from PCR product, bypassing the usual need to know the primer sequence. Lack of abundance of primers derived from a PCR product is compensated for by the incorporation of an arbitrary 5'TAG sequence which acts as a surrogate template target for the bulk amplification phase. We use the technique to amplify clonospecific rearranged immunoglobulin genes, which have applications as markers of lymphoid neoplasms for tracing the success of therapy. The principle may have wider application wherever conserved and variable regions of DNA are juxtaposed.