Single Polymerase Research Articles

Association of S100B polymorphisms and serum S100B with risk of systemic lupus erythematous in a Chinese population.

The aim of this study was to investigate whether the S100B polymorphisms are associated with systemic lupus erythematous (SLE) in a Chinese population. A total of 313 SLE patients and 396 control subjects were enrolled in the present study. The genotypes of three SNPs (rs9722, rs881827 and rs1051169) in S100B gene were detected by single base extension polymerase chain reaction (SBE-PCR). Serum S100B levels were determined by enzyme-linked immunosorbent assay (ELISA). Rs1051169 was associated with an increased risk of SLE (C vs. G: adjusted OR=1.46, 95% CI, 1.18-1.80, p=0.001; CC vs. GG: adjusted OR=1.99, 95% CI, 1.32-3.02, p=0.001; CC+GC vs. GG: adjusted OR=1.54, 95% CI, 1.13-2.11, p=0.007; CC vs. GC+GG: adjusted OR=1.67, 95% CI, 1.16-2.42, p=0.006). Haplotype analysis showed that the G-G-C haplotype was associated with an increased risk of SLE (OR=1.50, 95% CI, 1.14-1.98, p=0.004). Stratified analyses showed that the rs1051169 polymorphism was associated with an increased risk of neurologic disorder in SLE patients (C vs. G: OR=1.78, 95% CI, 1.22-2.59, p=0.003; GC vs. GG: OR=2.33, 95% CI, 1.14-4.77, P=0.019; CC vs. GG: OR=3.02, 95% CI, 1.39-6.53, p=0.004; CC+GC vs. GG: OR=2.57, 95% CI=1.31-5.04, p=0.005). In addition, SLE patients with neurologic disorder carrying the rs1051169 GC/CC genotypes present a higher serum S100B levels compared with that carrying the GG genotype (p < 0.05). Our results indicate that the rs1051169 polymorphism may be involved in the pathogenesis of SLE.

12-plex highly polymorphic microsatellite marker set for parentage analysis in Banija spotted pigs

Microsatellites (MS) have been, for the last two decades, widely used for parentage analysis in all types of livestock, including pigs. Conservation efforts on Banija spotted pigs included genetic characterization of the breed with MS markers. Since recent comparison of pedigree and MS data revealed some inconsistencies, the aim of this study was to develop a set of highly polymorphic and heterozygous MS markers, which could be used for parentage analysis and to prevent pedigree errors. 12 MS markers with the polymorphic information content (PIC) above 0.62 were chosen and combined into a single multiplex polymerase chain reaction (PCR). The combined non-exclusion probability for one candidate parent (NE-1P) was 0.00246149, the combined NE-1P given the genotype of a known parent of the opposite sex (NE-2P) was 0.00003739 and combined non-exclusion probability for a candidate parent pair (NE-PP) was 0.00000003. Due to a high information content of selected MS markers it was possible to obtain high accuracy in parentage assignment, which was confirmed by analyzing actual data with known genetic relationships

Loss-of-Function Mutations in HLA-Class I Alleles in Acquire Aplastic Anemia: Evidence for the Involvement of Limited Class I Alleles in the Auto-Antigen Presentation of Aplastic Anemia

[Background] Acquired aplastic anemia (AA) is a rare syndrome characterized by pancytopenia and bone marrow hypoplasia. The cytotoxic T lymphocyte (CTL) attack against autologous hematopoietic stem progenitor cells (HSPCs) is thought to be responsible for bone marrow failure in the majority of AA cases; however, little is known about the target antigens of the CTLs. HLA class I-allele lacking leukocytes (HLA-LL) due to copy-number neutral loss of heterozygosity in the short arm of chromosome 6 (6pLOH) or somatic loss-of-function mutations in HLA class I genes are detected in approximately 20% of patients with newly diagnosed AA, and the presence of HLA-LL represents compelling evidence to support that CTLs specific to HSPCs are involved in the development of AA. Our recent studies using single nucleotide polymorphism array (SNP-A) genotyping and droplet digital polymerase chain reaction (ddPCR) revealed that HLA-B*40:02 is the most frequently lost among all class I alleles that are lost as a result of 6pLOH (Zaimoku, et al. Blood 2017). Various somatic loss-of-function mutations in B*40:02 revealed by deep sequencing in the study substantiated the important role of HLA-B4002 in the autoantigen presentation of AA. However, in the other 6pLOH(+) AA patients who did not possess HLA-B4002, which accounted for 20% of the total AA cases involving patients possessing HLA-LL, the allele in the missing haplotype that was responsible for the autoantigen presentation was largely unknown because the lost fragment of chromosome 6p usually contained 2 or more HLA class I alleles. [Objectives/Methods] To identify class I alleles other than HLA-B*40:02 that are critically involved in the auto-antigen presentation of AA, we screened a total of 624 patients for the presence of HLA-LL using monoclonal antibodies specific to class I HLA alleles, SNP-A, and ddPCR, and performed targeted deep sequencing of HLA class I genes by using SeqCap EZ Choice pobes (Roche) and MiSeq sequencer (Illumina). The paired fractions, including granulocytes that lacked an HLA-A allele and granulocytes that retained the HLA-A allele, as well as CD3+ T cells, were sorted using monoclonal antibodies specific to HLA-A alleles with a BD FACSAria Fusion system (BD Biosciences), and were subjected to DNA extraction. All DNA samples of granulocytes and control cells (CD3+ T cells or buccal mucosa cells) were prepared for targeted deep sequencing. [Results] One hundred and fourteen patients were found to be positive for HLA-LL and 62 (54.4%) of the 114 HLA-LL(+) patients did not carry B*40:02 (severe, n=30; non-severe, n=32; male, n=38; female, n=24; median age, 62 [range, 6-93] years). Apart from B*40:02 (45.6%), A*02:06 (24.6%) was the second-most frequent HLA class I allele in the lost haplotype. The targeted deep sequencing of 20 patients with HLA-LL revealed 6pLOH alone in 11 patients, and somatic loss-of-function mutations plus 6pLOH in 9 patients; none of the patients were positive for somatic loss-of-function mutations alone. Of note, somatic loss-of-function mutations were found in only 5 alleles (A*02:06 in four, B*40:01 in two, B*40:03, A*31:01, and B*54:01 in one each) out of 27 different alleles contained in the lost haplotype. Among the 9 patients with somatic loss-of-function mutations, the median number of mutations per patient was 1 (range, 1-2); these included a missense mutation (n=1), frameshift deletions (n=3) and nonsense mutations (n=7) (Figure). Four patients had a breakpoint of 6pLOH in between the HLA-A and C loci; their lost alleles were A*02:06 (n=2) and A*31:01 (n=2), and the occurrence of 6pLOH in the four patients was therefore attributed to the two HLA-A alleles. Sixty-six percent of the HLA-LL(+) B*40:02(-) patients had at least one of the five alleles in the lost haplotypes. The frequencies of each “high risk” allele found in patients possessing HLA-LL are summarized in Table. [Conclusions] In addition to B*40:02, five class I alleles including HLA-A*02:06, A*31:01, B*54:01, B*40:03 and B*40:01 are thought to play an essential role in the auto-antigen presentation by the HSPCs of Japanese AA patients. The frequencies of the six class I alleles in general Japanese population are much higher than those in the general Caucasian populations but similar to the frequencies in East Asian populations. The higher frequencies of the six alleles in comparison to Caucasian countries may account for the higher incidence of AA in East Asia. [Display omitted] DisclosuresTakamatsu:Ono: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen: Honoraria; Celgene: Honoraria, Research Funding. Nakao:Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria.

Molecular detection of genes encoding resistance to tetracycline and determination of plasmid-mediated resistance to quinolones in avian pathogenic Escherichia coli in Sukabumi, Indonesia.

Aim:This study aimed to identify genes encoding resistance to tetracycline (TE) and plasmid-mediated resistance to quinolones in Escherichia coli isolates from clinical cases of avian colibacillosis in Sukabumi, Indonesia.Materials and Methods:A total of 25 E. coli archive isolates were collected in 2013-2017 from clinical cases of avian colibacillosis in Sukabumi, Indonesia. All isolates were tested for TE and quinolone resistance using the disk diffusion method. TE -resistant E. coli isolates were screened for the presence of tet(A) and tet(B) genes by single polymerase chain reaction (PCR). The qnr(A), qnr(B), and qnr(S) genes were detected by multiplex PCR in quinolone-resistant E. coli isolates.Results:Result of this study shows that 19 of 25 (76%) E. coli isolates are resistant to oxytetracycline and 64% are resistant to TE; among them, 63.2% and 31.5% were positive tet(A) and tet(B), respectively. 13 out of 25 (52%) are resistant to ciprofloxacin and 36% are resistant to enrofloxacin either norfloxacin; among them, 61.6% were positive qnr(A), 7.7% were positive qnr(B), 23% were positive qnr(S), and 7.7% were positive both of qnr(A) and qnr(S).Conclusion:This study shows that a few pathogens of E. coli are resistant to TE and quinolone. The frequency of tet and qnr genes that are responsible for this resistance among avian pathogenic E. coli isolates in Sukabumi, Indonesia, was high.

Tracking DNA Synthesis with Single-Molecule Strand Displacement.

We have previously shown that double-stranded DNA labeled with a periodic series of fluorescent dyes can be used to track a single helicase. Here we demonstrate how this technique can be adapted to follow processive DNA synthesis. By monitoring strand displacement, we track the motion of a single ϕ29 DNA polymerase without labeling or altering the enzyme or the template strand, and without applying any force. We observe a wide range of speeds, with the highest exceeding by several times those observed in earlier in vitro single-molecule experiments. Because this method enables repeated observations of the same polymerase traversing identical segments of DNA, it should prove useful for determining the effects of sequence on DNA replication and transcription. In addition, future measurements of this type may allow us to examine in detail the interactions of individual DNA polymerases with other components of the replisome.

Epidemiological Survey of Thalassemia in Women of childbearing age in Quanzhou Area of Fujian Province in China

To investigate the gene prevalence and spectrum of thalassemia in the women of childbearing age in quanzhou area. Venous blood of the women were collected for study, all subjects were registered in each county of quanzhou area by using cluster sampling. Both the mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were used for screening thalassemia．Genotyping of the screened positive samples was performed by gap single polymerase chain reaction (gap-PCR) and reverse dot blot (RDB) hybridization．Unknown positive samples were analyzed with DNA sequencing. Out of all 7 082 samples, Three hundred and eighty four were identified as thalassemia gene carriers with a carrying rate of 5.42 %. The α and β thalassemia were 3.21% and 2.15% respectively. --SEA /αα was the most common genotype with 68.72 % in mutation types of α thalassemia, In addition gene, 2 cases of --THAI/αα and 1 case of αα/αααanti3.7 were also detected. IVS-Ⅱ-654/N and CD41-42/N were the most common gentypes with 75.00 % in mutation types of β thalassemia gene, 5 cases were found to be α β compogite thalassemia. The carrying rate of thalassemia gene in quanzhou area is higher, and with the most common gentypes including --SEA /αα、IVSⅡ-654(C→T)/N and CD41-42(-TTCT)/N. The study results are beneficial for the screening of thalassemia in the genetic consultation and the prenatal gene diagnosis.

A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases

Insertional RNA editing has been observed and characterized in mitochondria of myxomycetes. The single subunit mitochondrial RNA polymerase adds nontemplated nucleotides co-transcriptionally to produce functional tRNA, rRNA and mRNAs with full genetic information. Addition of nontemplated nucleotides to the 3′ ends of RNAs have been observed in polymerases related to the mitochondrial RNA polymerase. This activity has been observed with T7 RNA polymerase (T7 RNAP), the well characterized prototype of the single subunit polymerases, as a nonspecific addition of nucleotides to the 3′ end of T7 RNAP transcripts in vitro. Here we show that this novel activity is an editing activity that can add specific ribonucleotides to 3′ ends of RNA or DNA when oligonucleotides, able to form intramolecular or intermolecular hairpin loops with recessed 3′ ends, are added to T7 RNA polymerase in the presence of at least one ribonucleotide triphosphate. Specific ribonucleotides are added to the recessed 3′ ends through Watson-Crick base pairing with the non-base paired nucleotide adjacent to the 3′ end. Optimization of this activity is obtained through alteration of the lengths of the 5′-extension, hairpin loop, and hairpin duplex. These properties define a T7 RNAP activity different from either transcriptional elongation or initiation.

A new method to quantify atmospheric Poaceae pollen DNA based on the trnT-F cpDNA region

Abstract Background Pollen, mold spores, bacteria and viruses are the main biological substances in the atmosphere causing allergic symptoms and disease. Distinguishing pollen and spores is quite time consuming and requires a trained expert. There is a different approach to identification of these substances such as microscopic analysis. However, DNA based identification of these is becoming popular recently. Objective We evaluated the correlation between the quantity of DNA, which was amplified using trnT-F cpDNA specific primers in samples obtained from a high volume air sampler (HVAS), and concentration of Poaceae pollen collected with a Burkard trap. Materials and methods Here, we present a method for identifying and quantifying airborne Poaceae pollen using a single step polymerase chain reaction (PCR) technique. Forty daily air samples were collected by HVAS. The method was optimised using two different methods (M1 and M2) and the trnT-F cpDNA region was amplified using a Poaceae specific primer pair. The correlation between the quantity of DNA and pollen concentration was tested using R statistical programming language. Results Although a significant correlation was obtained between the M1 and M2 methods (R2=0.655, p&lt;0.01), the M2 method was more correlated with pollen concentration. The correlation between pollen and DNA content changed due to episodes that were observed during the pollen season. DNA concentrations from the PCR data were significantly correlated with pollen concentrations determined by light microscopy (R2=0.767, p&lt;0.01) in episode II using the M2 method and during the entire season (R2=0.469, p&lt;0.01) using M2. Conclusions The M2 method correctly identified Poaceae pollen in mixed air samples from Zonguldak Province. The non-coding trnT-F cpDNA region was used for the first time in aerobiological samples to identify Poaceae pollen. Use of this method that does not require DNA extraction may be a crucial step for real-time pollen monitoring devices to be developed in the future. The correlation strength between pollen and amplified DNA content could be improved using a sampler that has a lower absorption rate, and a more sensitive technique, such as qPCR.

A Single Nucleoside Viral Polymerase Inhibitor Against Norovirus, Rotavirus, and Sapovirus-Induced Diarrhea.

A safe and highly efficient antiviral is needed for the prophylaxis and/or treatment of viral diarrhea. We here demonstrate the in vitro antiviral activity of four 2'-C-methyl nucleoside analogues against noro-, rota-, and sapoviruses. The most potent nucleoside analogue, 7-deaza-2'-C-methyladenosine, inhibits replication of these viruses with a 50% effective concentration < 5 µM. Mechanistically, we demonstrate that the 2'-C-methyl nucleoside analogues act by inhibiting transcription of the rotavirus genome. This provides the first evidence that a single viral-diarrhea-targeted treatment can be developed through a viral-polymerase-targeting small molecule.

Effects of silver sulfide quantum dots coated with 2-mercaptopropionic acid on genotoxic and apoptotic pathways in vitro

Quantum dots (QDs) are highly promising nanomaterials in bioimaging system because of their bright fluorescence, broad UV excitation, narrow emission band, and high photostability. Recently, there is a great activity on Ag2S quantum dots for both imaging and drug/gene delivery due to the potential of having a better cytocompatability and near infrared luminescence. 2-Mercaptopropionic acid (2 MP A)-coated silver sulfide (Ag2S) QDs were reported as the most luminescent, stable, anionic Ag2S QDs in the literature. In this study, we aim to determine the cytotoxicity of 2 MP A/Ag2S in Chinese hamster lung fibroblast (V79) cells. The genotoxic and apoptotic effects of 2 MP A/Ag2S QDs were assessed by the alkaline single cell electrophoresis assay and real time polymerase chain reaction techniques, respectively. The cell viability decreased above 200 μg/ml and 800 μg/ml for MTT tetrazolium and neural red uptake assays, respectively. DNA damage was not observed by 2 MP A/Ag2S QDs at the studied concentration levels (5–2000 μg/ml). The levels of mRNA expression of p53, caspase 3, caspase 9, bax, bcl-2, survivin were not changed by 2 MP A/Ag2S QDs below IC50 (around 1000 μg/ml). Hence, 2 MP A/Ag2S QDs did not show any cytotoxic or genotoxic effects in V79 cells at lower doses. We conclude that the biocompatibility of 2 MP A/Ag2SODs makes them suitable for cell labeling applications.

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp.

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

Toll-like receptor expression in pulmonary sensory neurons in the bleomycin-induced fibrosis model.

Airway sensory nerves are known to express several receptors and channels that are activated by exogenous and endogenous mediators that cause coughing. Toll-like receptor (TLR) s are expressed in nociceptive neurons and play an important role in neuroinflammation. However, there have been very few studies of TLR expression in lung-derived sensory neurons or their relevance to respiratory symptoms such as cough. We used the bleomycin-induced pulmonary fibrosis model to investigate the change in TLR expression in pulmonary neurons and the association of TLRs with transient receptor potential (TRP) channels in pulmonary neurons. After 2 weeks of bleomycin or saline administration, pulmonary fibrosis changes were confirmed using tissue staining and the SIRCOL collagen assay. TLRs (TLR 1–9) and TRP channel expression was analyzed using single cell reverse transcription polymerase chain reaction (RT-PCR) in isolated sensory neurons from the nodose/jugular ganglion and the dorsal root ganglion (DRG). Pulmonary sensory neurons expressed TLR2 and TLR5. In the bleomycin-induced pulmonary fibrosis model, TLR2 expression was detected in 29.5% (18/61) and 26.9% (21/78) of pulmonary nodose/jugular neurons and DRG neurons, respectively. TLR5 was also detected in 55.7% (34/61) and 42.3% (33/78) of pulmonary nodose/jugular neurons and DRG neurons, respectively, in the bleomycin-induced pulmonary fibrosis model. TLR5 was expressed in 63.4% of TRPV1 positive cells and 43.4% of TRPM8 positive cells. In conclusion, TLR2 and TLR5 expression is enhanced, especially in vagal neurons, in the bleomycin-induced fibrosis model group when compared to the saline treated control group. Co-expression of TLR5 and TRP channels in pulmonary sensory neurons was also observed. This work sheds new light on the role of TLRs in the control and manifestation of clinical symptoms, such as cough. To understand the role of TLRs in pulmonary sensory nerves, further study will be required.

Streptococcus pneumoniae Serotyping by a Single Polymerase Chain Reaction–Based Multiplex Assay

Background Streptococcus pneumoniae is a prominent pathogen in children younger than 5 years as well as elderly people. Capsular serotyping of S. pneumoniae is necessary to develop the new vaccines and prevent invasive and noninvasive infections by S. pneumoniae. In this study, we used 2-step multiplex polymerase chain reaction (mPCR) that contained primers to detect PCV13 (13-valent pneumococcal conjugated vaccine) and non-PCV13 serotypes in different clinical and normal flora samples. Methods A total of 100 S. pneumoniae isolates were obtained between 2013 and 2015 in Tehran, Iran. The sources of isolates were clinical and normal flora. Clinical isolates were eye infection (26%), blood (19%), sputum (18%), sinusitis and cerebrospinal fluid (9% each), trachea (7%), pleural aspirate (3%), otitis (3%), and urine, bronchoalveolar lavage, and abscess (2% each). Moreover, 43 normal flora isolates were collected from healthy individuals. The strain isolates were tested for antimicrobial susceptibility and serotyped by mPCR. Results The highest rate of resistance was seen for trimethoprim-sulfamethoxazole (96%) followed by tetracycline (77%), erythromycin (64%), clindamycin (56%), chloramphenicol (44%), and penicillin (26%). All isolates were susceptible to imipenem, ceftriaxone, vancomycin, linezolid, gemifloxacin, levofloxacin, moxifloxacin, and ofloxacin. By using mPCR, 91 and 7 isolates were typed in the first and second reactions, respectively. Two isolates were identified as nontypeable. The most frequent serotypes in 98 typeable serotypes were 23F (n = 21 [22%]), 14 (n = 19 [20%]), 3 (n = 13 [13%]), and 19F (n = 13 [13%]). Conclusions Our multiplex assay is a precise and reliable method that can be used instead of the Quellung reaction for S. pneumoniae serotyping studies.

High CDKN2A/p16 and Low FGFR3 Expression Predict Progressive Potential of Stage pT1 Urothelial Bladder Carcinoma.

A recent study on the comprehensive genomic profile of advanced urothelial bladder cancer (UBC) showed cyclin-dependent kinase inhibitor 2A (CDKN2A) and fibroblast growth factor receptor 3 (FGFR3) as the most often clinically relevant genomic alterations. Therefore, the prognostic role of FGFR3 and CDKN2A/p16 for pT1 UBC was studied. Clinical data and formalin-fixed paraffin-embedded tissues of pT1 UBC treated with an organ-preserving approach was analyzed retrospectively. Total RNA was isolated using commercial RNA extraction kits and mRNA expression of CDKN2A/p16 and FGFR3 was measured using single step reverse transcription quantitative real time polymerase chain reaction using RNA-specific TaqMan assays. Data from 296 patients (79.4% male; median age: 72 years) could be used for the final evaluation. Spearman correlation revealed a statistically significant negative correlation between mRNA expression of CDKN2A/p16 and FGFR3. There was a positive correlation between CDKN2A/p16 and G3 tumors (ρ= 0.1875; P= .0012) and associated carcinoma in situ (ρ= 0.1703, P=.0033) and a negative correlation between FGFR3 and these factors (ρ=-0.2791, P< .0001 and ρ=-0.2182, P=.0002). High CDKN2A/p16 expression (≥38.04) and low FGFR3 expression (<39.14) were statistically significantly associated with worse progression-free survival (PFS; P= .0194 and P= .0089). Multivariate Cox regression analysis could identify patients with low FGFR3 and high CDKN2A/p16 expression (log rank (LR) χ2= 10.69; P=.0048) as well as tumor size≥3 cm (LR χ2= 6.03; P= .0141) as independent predictors for PFS. High expression of CDKN2A/p16 and low expression of FGFR3 show a correlation with established prognostic features for non-muscle-invasive bladder cancer and can predict progression of stage pT1 UBC.

Single Subunit RNA Polymerases: An Insight into their Active Sites and Catalytic Mechanism

Single Subunit RNA Polymerases: An Insight into their Active Sites and Catalytic Mechanism

Two Rapid Methods to Identify Three Species of Pathogenic Vibrio in Penaeus vannamei

Vibrio species frequently infect Penaeus vannamei causing great economic losses to the Whiteleg shrimp industry. Rapid detection of pathogenic Vibrio infection would improve the fight against these diseases. In this study, single and multiple polymerase chain reaction (PCR) methods were developed to detect three species of pathogenic Vibrio: Vibrio fluvialis, Vibrio anguillarum, and Vibrio alginolyticus. Specific primers were designed for the toxR gene of V. fluvialis, the flaA gene of V. anguillarum, and the pyrH gene of V. alginolyticus. The bacteria were used as templates to establish a 25 μL reaction system for PCR amplification. The results showed that single and specific PCR amplification products of expected sizes were obtained (228bp, 1665bp, and 383bp, respectively). The lowest concentration detected for the three Vibrio species were 5.21×102 , 2.70×104 , and 2.48×102 colony forming units (cfu)/mL, respectively. We also developed a multiplex PCR method to identify the three Vibrio species accurately, and with improved identification efficiency. In addition, quantitative real-time PCR (qPCR) was used to identify the minimum detectable DNA concentration for the three Vibrio species (1.0×10-6 nmol/L for V. fluvialis, 1.0×10-7 nmol/L for V. anguillarum, and 1.0×10- 8 nmol/L for V. alginolyticus). Technical requirements for ordinary PCR are low, therefore PCR is a feasible technique to detect and diagnose Penaeus vannamei bacterial disease.

A Novel Multiplex PCR-RFLP Method for Simultaneous Genotyping of CYP3A4*4 A>G, CYP3A4*18B G>A and CYP3A4*22 C>T.

BackgroundCytochrome P450 3A enzymes exhibit a variety of physiological roles and have been reported to be the most predominant enzymes involved in drugs metabolism. Single nucleotide polymorphisms (SNPs) in the genes that code for these enzymes may result in functional changes that affect enzyme activity. CYP3A4 is an important enzyme in the metabolism of many important drugs used in the treatment of breast cancer.MethodsA total of 94 post-menopausal breast cancer patients were recruited for the study and their DNA was isolated for polymerase chain reaction (PCR). The primers were designed using Primer3 software with primer specificities checked via the Basic Local Alignment Tool (BLAST) database. The primer specificity, functionality and annealing temperature were first investigated using uniplex PCR protocols, followed by a single multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The digested amplification fragments were analysed by gel electrophoresis and subsequently validated by sequencing.ResultsA multiplex PCR-RFLP method was successfully developed for simultaneous detection of CYP3A4*4, CYP3A4*18B and CYP3A4*22 in a population of post-menopausal breast cancer patients.ConclusionThe technique is simple, cost-effective, time-saving and can be routinely applied in the identification of SNPs and determination of allelic and genotypic frequencies of CYP3A4*4, CYP3A4*18B and CYP3A4*22.

Assessing S. mansoni prevalence in Biomphalaria snails in the Gombe ecosystem of western Tanzania: the importance of DNA sequence data for clarifying species identification

BackgroundSnails are essential for the transmission and maintenance of schistosomiasis in endemic areas, as they serve as intermediate hosts for schistosome parasites. A clear understanding of the snail species present, their local distribution and infection status is therefore a prerequisite for effective control of schistosomiasis. The purpose of this study was to establish the infection status and distribution of Schistosoma mansoni in snails in the Gombe area along the shores of Lake Tanganyika in western Tanzania, using both detection of cercarial shedding and molecular approaches.MethodsSnails were collected from streams located close to human settlements in Gombe National Park, as well as from nearby villages (Kiziba, Mtanga, Mwamgongo and Bugamba) and the largest town in the region (Kigoma). Snails were individually exposed to light to induce shedding of schistosome larvae, which were examined using a compound light microscope. Additionally, the internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster was simultaneously amplified in both snails and their trematodes using a single polymerase chain reaction (PCR) and sequenced to confirm species identification.ResultsSnails morphologically identified as Biomphalaria pfeifferi were present in all streams except at Mtanga but their distribution was patchy in both time and space. Sequencing of PCR products indicated that not all snails were B. pfeifferi. None of the snails from Gombe or Bugamba shed schistosome larvae, while larvae were shed at all other sites. Overall, an infection prevalence of only 12% was observed in snails based on cercarial shedding. While 47% of the snails were PCR-positive for the 500 bp ITS fragment, which was predicted to indicate infection with S. mansoni, sequence data demonstrated that these bands are not species-specific and can be amplified from other trematode infections. In addition, a 1000 bp band was amplified in 14% of samples, which was identified as a trematode in the family Derogenidae.ConclusionsThe results support the previous assumption that B. pfeifferi snails may be involved in transmitting schistosomiasis in the area but suggest that the community structure of both snails and trematodes may be more complicated than previously thought. This emphasises the importance of confirming species identifications using sequencing, rather than relying only on PCR-based diagnostics or cercarial shedding.

Influence of DNA Lesions on Polymerase-Mediated DNA Replication at Single-Molecule Resolution.

Faithful replication of DNA is a critical aspect in maintaining genome integrity. DNA polymerases are responsible for replicating DNA, and high-fidelity polymerases do this rapidly and at low error rates. Upon exposure to exogenous or endogenous substances, DNA can become damaged and this can alter the speed and fidelity of a DNA polymerase. In this instance, DNA polymerases are confronted with an obstacle that can result in genomic instability during replication, for example, by nucleotide misinsertion or replication fork collapse. It is important to know how DNA polymerases respond to damaged DNA substrates to understand the mechanism of mutagenesis and chemical carcinogenesis. Single-molecule techniques have helped to improve our current understanding of DNA polymerase-mediated DNA replication, as they enable the dissection of mechanistic details that can otherwise be lost in ensemble-averaged experiments. These techniques have also been used to gain a deeper understanding of how single DNA polymerases behave at the site of the damage in a DNA substrate. In this review, we evaluate single-molecule studies that have examined the interaction between DNA polymerases and damaged sites on a DNA template.

SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing

CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications. Typically, sgRNA is constitutively expressed relying on RNA polymerase III promoters. Polymerase II promoters initiate transcription in a flexible manner, but sgRNAs generated by RNA polymerase II promoter lost their nuclease activity. To express sgRNAs in a tissue-specific fashion and endow CRISPR with more versatile function, a novel system was established in a polycistron, where miRNAs (or shRNAs) and sgRNAs alternately emerged and co-expressed under the control of a single polymerase II promoter. Effective expression and further processing of functional miRNAs and sgRNAs were achieved. The redundant nucleotides adjacent to sgRNA were degraded, and 5′- cap structure was responsible for the compromised nuclease capacity of sgRNA: Cas9 complex. Furthermore, this strategy fulfilled conducting multiplex genome editing, as well as executing neural- specific genome editing and enhancing the proportion of homologous recombination via inhibiting NHEJ pathway by shRNA. In summary, we designed a new construction for efficient expression of sgRNAs with miRNAs (shRNAs) by virtue of RNA polymerase II promoters, which will spur the development of safer, more controllable/regulable and powerful CRISPR/Cas9 system-mediated genome editing in a wide variety of further biomedical applications.