Multiplexed Detection of Anthrax-Related Toxin Genes

The specificity and sensitivity of polymerase chain reaction (PCR) primers developed for 'Candidatus Liberibacter solanacearum' and 'Candidatus Liberibacter psyllaurous' were evaluated in conventional and real-time PCR assays. All PCR primers were specific for 'Ca. L. psyllaurous' and 'Ca. L. solanacearum' insomuch as they did not detect other prokaryotic plant pathogens that affect potato except for the putative pathogens associated with psyllid-yellows and haywire. Conventional PCR assays were capable of detecting 0.19 to 1.56 ng of total DNA per reaction, and real-time PCR was found capable of detecting 1.56 to 6.25 ng of total DNA per reaction, depending on the specific PCR primer set used. 'Ca. Liberibacter' species associated with zebra complex disease (ZC) was confirmed in plants affected by this disease throughout Texas from 2005 to 2008, in seed tubers produced in Wyoming in 2007, and in Colorado, Kansas, Nebraska, and Mexico in 2008. A multiplex PCR assay using 'Ca. L. solanacearum'-specific primers and primers specific for the β-tubulin DNA regions from potato was developed, providing possible utility of the multiplex assay for 'Ca. Liberibacter' detection in different solanaceous plant species. Preliminary studies suggest silverleaf nightshade (Solanum elaeagnifolium), wolfberry (Lycium barbarum), black nightshade (S. ptychanthum), and jalapeno pepper (Capsicum annuum) as additional solanaceous hosts for the ZC-associated bacterium. The 'Ca. Liberibacter' species detected in all samples divided into two clusters sharing similarity of 99.8% in their partial 16S rRNA gene sequences and 99.3% in their partial intergenic spacer region (ISR)-23S rRNA gene sequences. Genetic variation in the 16S rDNA region consistently matched that of the ISR-23S rDNA region. In this partial 16S-ISR-23S rDNA region, there was a total of eight single nucleotide polymorphisms among 'Ca. L. psyllaurous' and 'Ca. L. solanacearum' "strains" investigated in this study. 'Ca. L. solanacearum' and 'Ca. L. psyllaurous' were shown to be very closely related bacteria, if not the same, by successful amplification using a combination of forward primer of 'Ca. L. solanacearum' and reverse primer of 'Ca. L. psyllaurous' in ZC-affected potato samples. This finding clarifies the current taxonomic status of 'Ca. L. solanacearum' and 'Ca. L. psyllaurous'. The detection of 'Ca. L. solanacearum' from haywire-symptomatic potato samples demonstrates that this bacterium might also be associated with this disease.

The goal of this research was to develop a new genetic database of simple sequence repetition (SSR) primers for faba and classify them according to their target genes and respective biological processes. Approximately 75,605 and 148,196 previously published genomic and transcriptomic faba sequences, respectively, have been used to detect possible SSRs. The numbers of identified SSRs from each dataset were 25,502 and 12,319, respectively. The distribution of different repeat classes indicated that trinucleotides represent the largest number of repeat counts, followed by dinucleotides. The extracted genic SSR sequences were used to design 1091 polymerase chain reaction (PCR) primers, of which only 238 (21.8%) primers target genomic sequences and the other 853 PCR primers targeted transcriptomic sequences. The annotation of gene-targeted SSRs showed that approximately 897 genes were targeted by our SSR primers. Approximately 1890 gene ontology (GO) identification codes have been obtained. The GO keywords were distributed among distinct molecular cell features. The highest redundancies involved 554 technical words, 196 domains, and 160 molecular feature phrases. These GO codes belonged to the general level of GO and included molecular function, cellular component, and biological process (544, 670, and 676 GOs, respectively). Twenty-seven SSR PCR primers were synthesized to 12 Egyptian faba bean genotypes. Approximately 11 SSR provided one to two PCR bands, whereas other SSRs provided only one sharp band with polymorphic band size. There were 13 polymorphic primers. The polymorphism information content was 0.3, which implied moderate informativeness.

Objective To investigate the relationship between NINJ2 gene polymorphism and stroke, and the differences of serum levels of tumor necrosis factor-αt (TNF-α), NGF, interleukin-6 (IL-6)and P-Selectin in healthy controls and patients under recovery stage. Methods Fifty-two patients with large-artery atherosclerosis (LAA) infarction, 85 patients with small-artery occlusion lacunar (SAO)infarction, 50 patients with intracerebral hemorrhage (ICH) and 66 healthy controls were included in this study. Genotypes of the 2 single nucleotide polymorphism (SNP) sites (rs12425791 and rs11833579) in NINJ2 gene were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RLFP) method. The differences of genotypes and alleles frequencies of the 2 SNP sites between each 2 different groups were analyzed and compared. Multinomial logistic regression was used to calculate the odds ratio (OR) of genotypes in each patient group, and 95% confidential interval (95% CI)was given. The serum levels ofTNF-α, NGF, IL-6 and P-Selectin were tested by ELISA method, and compared between groups and within group classified by genotypes. Results In regard to rs12425791 site, the frequencies of AG and AA+AG genotypes in LAA and SAO groups were significantly higher than those in control group (P＜0.05), while this difference was not found between the ICH group and control group (P＞0.05); the frequency of A allele in the SAO group was significantly higher than that in the control group (P＜0.05), while this difference was not found between the control group and both the LAA and ICH groups (P＞0.05). In regard to rs11833579 site, no significant differences in the genotypes and alleles were noted between all the patient groups and control group (P＞0.05). After adjusting the influence of other risk factors, the multinomial logistic regression analysis showed that the onset of stroke was still significantly associated with the AG genotype at rs12425791 site in the LAA group (OR=4.298,95%CI=1.430-12.922) and AG, AG+AA genotypes at rs12425791 site in the SAO group (OR=3.923 and 2.937, 95%CI= 1.417- 10.860 and 1.119-7.710). Neither genotypes in rs 11833579 site were significantly associated with the onset of stroke. No significant differences of serum levels of TNF-α, NGF, IL-6 and P-Selectin were noted between each patient group under recovery stage and control group (P＞0.05); in regard to rs12425791 site, the serum level of TNF in LAA group with different genotypes was significantly different (P＜0.05) and the serum level of P-Selectin in ICH group with different genotypes was significantly different (P＜0.05); in regard to rs11833579 site, the serum level of NGF in LAA group with different genotypes was statistically different (P＜0.05). Conclusion This SNP site (rs12425791)is significantly associated with ischemia stroke and the A allele increases the risk of being susceptible to this disease in Chinese Han population. That SNP site (rs1 1833579) is not significantly associated with stroke. No significant differences of TNF-α, NGF, IL-6, P-Selectin serum levels are noted between patients under recovery stage and controls. Key words: NINJ2 gene; Polymorphism; single nucleotide; Stroke; Tumor necrosis factor-α; NGF; Interleukin-6; P-Selectin

Identification of Functional Genetic Variants in and Their Association With Risk of Esophageal Cancer

These findings indicate that genetic variants in COX-2 may play a role in mediating susceptibility to esophageal cancer.

Locked Nucleic Acids Can Enhance the Analytical Performance of Quantitative Methylation-Specific Polymerase Chain Reaction

Novel Functional Single Nucleotide Polymorphisms in the Latent Transforming Growth Factor-β Binding Protein-1L Promoter: Effect on Latent Transforming Growth Factor-β Binding Protein-1L Expression Level and Possible Prognostic Significance in Ovarian Cancer

Several published polymerase chain reaction (PCR) primers to identify Pseudomonas syringae pv. actinidiae , the causal organism of bacterial canker of kiwifruit, were found not to be specific. Two new sets of PCR primers, PsaF1/R2 and PsaF3/R4, were designed to be complementary to a portion of the 16S–23S rDNA intertranscribed spacer (ITS) regions. These primers amplified a DNA fragment from strains of P. syringae pv. actinidiae , but not from 56 strains of bacteria from six genera and 17 species, except for a strain of the tea pathogen, P. syringae pv. theae . When tested against DNA extracted from a further 20 strains from Japan, Korea, Italy and the USA deposited in culture collections as P. syringae pv. actinidiae , all except six cultures produced the expected product of 280 bp with PsaF1/R2 and 175 bp with PsaF3/R4. Results of multilocus sequence analysis using five housekeeping genes ( gyrB , acnB , rpoD , pgi and cts ) showed that none of these six strains was phylogenetically similar to P. syringae pv. actinidiae. In contrast to the P. syringae pv. actinidiae type strain, these strains were positive in the determinative tests for ice nucleation and syringomycin production. It is suggested that these six strains were incorrectly identified as P. syringae pv. actinidiae . It was not possible to distinguish P. syringae pv. actinidiae from the phylogenetically similar P. syringae pv. theae using the ITS, gyrB , acnB , rpoD , pgi or cts gene regions to design PCR primers. Because P. syringae pv. theae is unlikely to be found on kiwifruit, primers PsaF1/R2 and PsaF3/R4 are recommended for screening bacteria isolated from kiwifruit tissue.

Optimal pcr primers for rapid and accurate detection of Aspergillus flavus isolates

Rapid advances in DNA sequencing technologies have made it increasingly cost-effective to obtain accurate and timely large-scale genomic sequence data on individuals (short read massively parallel or next generation [next-gen]). A next-gen molecular diagnostic approach that has seen rapid deployment in the clinic over the last year is exome sequencing. Whole exome sequencing covers all protein-coding genes in the genome (≈1.1% of genome), and an exome test for a single patient generates ≈6 gigabases (109 bp) of DNA sequence data. A key challenge facing routine use of next-gen data in patient diagnosis and management is data interpretation. What sequence variant findings are relevant to diagnosis (pathogenic mutations)? What sequence variant findings are relevant to clinical care but not necessarily to patient diagnosis (clinically actionable incidental data)? What sequence information should be stored, and where can it be stored? This review provides a tutorial on current approaches to answering these questions. A recent landmark study showed that application of next-gen sequencing to a large cohort of idiopathic dilated cardiomyopathy patients found ≈27% of patients to show mutations of the titin gene, the most complex gene in the genome (363 exons). We use titin in cardiomyopathy as an exemplar for explaining next-gen sequencing approaches and data interpretation. Decreasing sequencing costs and broad dissemination of next-generation (next-gen) equipment and expertise are increasing availability of massively parallel sequencing of patient DNA samples (short read massively parallel or next-gen sequencing).1,2 Most rapidly expanding is exome sequencing, where all protein-coding sequences (exons) are selected from total genomic DNA and selectively sequenced.3 Alternative approaches to next-gen sequencing include targeted sequencing (TS) and whole genome (complete genome) sequencing. Currently, marketed targeted Sanger sequencing panels using traditional individual exon-by-exon sequencing remain expensive and time consuming, and massively parallel next-gen approaches are beginning to supplant …

The visualization of gene structure plays an important role in polymerase chain reaction (PCR) primer design, especially for eukaryotic genes with a number of splice variants that users need to distinguish between via PCR. Here, we describe a visualized web server for primer design named VizPrimer. It utilizes the new information technology (IT) tools, HTML5 to display gene structure and JavaScript to interact with the users. In VizPrimer, the users can focus their attention on the gene structure and primer design strategy, without wasting time calculating the exon positions of splice variants or manually configuring complicated parameters. In addition, VizPrimer is also suitable for the design of PCR primers for amplifying open reading frames and detecting single nucleotide polymorphisms (SNPs). VizPrimer is freely available at http://biocompute.bmi.ac.cn/CZlab/VizPrimer/. The web server supported browsers: Chrome (≥5.0), Firefox (≥3.0), Safari (≥4.0) and Opera (≥10.0). zhangcg@bmi.ac.cn; yangyi528@vip.sina.com.

We have developed statistical models for estimating the failure rate of polymerase chain reaction (PCR) primers using 236 primer sequence-related factors. The model involved 1314 primer pairs and is based on more than 80 000 PCR experiments. We found that the most important factor in determining PCR failure is the number of predicted primer-binding sites in the genomic DNA. We also compared different ways of defining primer-binding sites (fixed length word versus thermodynamic model; exact match versus matches including 1–2 mismatches). We found that the most efficient prediction of PCR failure rates can be achieved using a combination of four factors (number of primer-binding sites counted in different ways plus GC% of the primer) combined into single statistical model GM1. According to our estimations from experimental data, the GM1 model can reduce the average failure rate of PCR primers nearly 3-fold (from 17% to 6%). The GM1 model can easily be implemented in software to premask genome sequences for potentially failing PCR primers, thus improving large-scale PCR-primer design.

Melting-curve procedures track DNA denaturation in real time and so provide an effective way of assessing sequence variants. Dynamic allele-specific hybridization (DASH) is one such method, based on fluorescence, which uses heat to denature a single allele-specific probe away from one strand of a polymerase chain reaction product attached to a solid support. DASH is a proven system for research genotyping, but here we evaluate it for DNA diagnostics under two scenarios. First, for mutation scanning (resequencing), a human genomic sequence of 97 bp was interrogated with 15 probes tiled with 12-base overlaps, providing up to fourfold redundancy per base. This test sequence spanned three high-frequency single nucleotide polymorphisms, all of which were correctly revealed in 16 individuals. Second, to score multiple different mutations in parallel, 18 alterations in the gyrA gene of Salmonella were assessed in 62 strains by using wild-type- and mutation-specific probes. Both experiments were performed in a blinded manner, and all results were confirmed by sequencing. All DNA variants were unambiguously resolved in every sample, with no false-positive or false-negative signals across all of the investigations. In conclusion, DASH performs accurately and robustly when applied to DNA diagnostic challenges, including mutation scoring and mutation scanning.

Fusarium oxysporum f. sp. fragariae is a fungal pathogen causing Fusarium wilt on strawberry. Polymerase chain reaction (PCR) primers that can discriminate F. oxysporum f. sp. fragariae from nonpathogenic F. oxysporum would greatly assist pathogen identification. In order to develop a molecular diagnostic tool for this pathogen, transposable elements in the pathogen were characterized and used for designing a specific set of PCR primers. Portions of the transposable elements Fot3, Han, Hop, Hornet1, and Skippy were detected in all 33 strains of F. oxysporum f. sp. fragariae tested by PCR, whereas Foxy was detected in 32 strains and Impala sequences were detected in 30 strains. Two types of sequences were detected for Hop, two types for Impala, and three types for Skippy. The genomic region between Han and Skippy was amplified by an inter-retrotransposon amplified polymorphism technique, and PCR primers (FofraF and FofraR) to specifically identify F. oxysporum f. sp. fragariae were designed from this region. The developed PCR primers discriminated F. oxysporum f. sp. fragariae strains from nonpathogenic F. oxysporum strains and five other formae speciales. Conidia of F. oxysporum f. sp. fragariae could be detected in brown lowland-type soil by PCR using the primers. After preculturing the soil sample on FoG2 medium, 1 × 102 conidia/g of soil could be detected; without preculturing, 1 × 103 conidia/g of soil were detected.