Chimeric Primers Research Articles

A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α-primase.

The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of Xenopus laevis polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5' end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer-template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase.

CRISPR/Cas12a cleavage-mediated isothermal amplification lights up the dimeric G-quadruplex signal unit for ultrasensitive and label-free detection of circulating tumor DNA

Circulating tumor DNA (ctDNA) is a valuable and potential cancer biomarker for real-time monitoring, and plays a vital role in cancer early diagnosis. In this work, we developed an ultrasensitive and label-free fluorescent biosensor for ctDNA detection. A well-designed RNA-DNA chimeric primer (RD) with phosphate groups modified at the 3-terminal was cleaved by target-initiated CRISPR/Cas12a, and then the subsequent exponential amplification reaction (EXPAR) was started to produce numerous G-rich sequences, which could be assembled with Thioflavin T to form a dimeric G-quadruplex (dimer-G4/ThT) and significantly enhance the fluorescence. Due to the innovative combination of the specificity of CRISPR/Cas12a and RD primers, the high amplification efficiency of EXPAR, and the strong fluorescence response of dimer-G4/ThT, this strategy demonstrated excellent sensitivity at the aM level. In addition, the sensing system showed satisfactory anti-interference ability and reliable accuracy in complex serum environment and tumor cell lysate. More importantly, this strategy avoided the complicated and expensive labeling process of traditional signal probes, making the construction of fluorescent biosensor simpler and more practical, offering practical insights for molecular diagnostics and clinical applications.

Universal primer multiplex PCR assay for detection and genotyping of porcine astroviruses

Porcine astroviruses (PAstV) are members of the family Astroviridae, Mamastravirus genus and have been identified to have five genotypes (PAstV1–5). These viruses are highly prevalent in pigs and can cause enteric disease as well as neurological or respiratory symptoms depending on their genotypes. At present, the epidemiological impacts of some PAstV genotypes on pigs are largely unknown and hence continuously monitoring of these PAstVs may be needed. The purpose of this research was to develop an improved and efficient detection tool for PAstVs and to evaluate the developed method using clinical samples. Initially, a set of five chimeric primers (CP), each comprising genotype specific primer pairs with an identical universal adapter at the 5’ end, and a universal primer (UP) that is identical to universal adapter sequence, were designed. With these tools in place, a novel multiplex PCR system with universal primer was established for the simultaneous detection of the five types of PAstV. This method can specifically detect PAstV genotypes, with a limit of detection (LOD) of 5 copies/μL for each genotype irrespective of single or mixed target template. Using this new assay, 273 pig fecal samples were investigated for further assay evaluation. Among all samples, the positive rate was 70.0% with PAstV4 in 56.8% of the samples, PAstV2 in 38.8%, PAstV1 in 16.8%, and PAstV5 in 11.0%. More than one PAstV in a sample were detected in 39.2% of the samples. The consistency rate between the novel multiplex PCR and singleplex PCRs was 96.4–100%. Given its rapidity, specificity and sensitivity, the novel multiplex PCR is a useful approach for demonstrating single or mixed genotype infections of PAstV.

A Robust Ex-Vivo Erythropoiesis System For Studying Transcriptional Regulation Of Globin Genes In Thalassaemia

Ex-vivo differentiation of hematopoietic stem cells and progenitors to erythroid cells helped tremendously in our understanding of transcriptional and epigenetic regulation of normal erythroid differentiation as large number of nucleated cells can be generated by these protocols. However, to study the disease mechanisms in haematological diseases, there are practical difficulties in obtaining mobilized CD34+ cells from the patients. Alternatively, peripheral blood mononuclear cells (PBMNCs), which have a small fraction of haematopoietic progenitors, have also been used to obtain cultured erythroid cells (cErCs). Though PBMNCs is an alternative source, the current protocols using these cells do not generate sufficient number of cErCs, which is a major hurdle for performing transcriptional and epigenetic studies in erythroid cells from patients with β thalassaemia and sickle cell disease. We established a modified two phase culture protocol using a serum free basal medium with IL-3, IL-6, Flt3, SCF in phase I and Epo, SCF, IL-3 and iron compounds in phase II and could generate up to 108 cells from PBMNCs isolated from 5-10ml blood of patients with homozygous β thalassaemia and those who were compound heterozygous for β thalassaemia and HPFH or δβ thalassaemia. Real time quantitative PCR (qPCR) analysis showed steady increase in the expression of α and β globin genes in different stages of erythropoiesis. As we obtained a large number of nucleated cells using this protocol, we could perform chromatin immunoprecipitation (ChIP) to study the binding of RNA Polymerase II (RNAPII), transcription cofactors, CBP and p300, and histone modifications at the regulatory sequences of β globin cluster. High levels of RNAPII was found at the coding sequences of β globin gene in the cErCs from both normal individuals and patients with splice site mutations suggesting that these mutations do not cause significant reduction in the transcription rate. Both CBP and p300 were found to bind at the HS3 and HS2 regions of locus control region in both normal and β thalassaemia cultures though they were not found at the promoter sequences of actively transcribed genes in the β globin cluster. In the patients compound heterozygous for β thalassaemia and HPFH-3 or (Aγδβ)° deletion/Inversion, we found increased γ globin gene expression compared to the normal controls and ChIP also showed high levels of RNAPII occupancy at the coding sequences of γ globin gene. As it has been shown earlier that alternative splicing is a transcriptional regulatory mechanism for cell specific expression of genes, we performed experiments to identify the alternatively spliced transcripts of β globin gene and their co-ordinated expression in human erythropoiesis. RT-PCRs and sequencing of β globin cDNA using various sets of primers that bind at the upstream, downstream and coding sequences showed 3 aberrantly spliced transcripts formed by the cryptic splice sites in exon1 and 4 novel transcripts by the activation of splice sites at the 5'UTR. Interestingly, 5' splice site mutations in intron 1 did not activate cryptic splice sites in the coding sequences and instead, they activated specific cryptic splice sites upstream b globin gene. Q-PCR with chimeric primers for the exact quantitation of the splice variants showed that the splice site mutations also cause significant reduction (> 90%) in expression of normally spliced transcripts suggesting that nonsense mediated decay is not the major mechanism for the reduced β globin production by these mutations. We found high levels of one of the aberrantly spliced transcripts with 5' splice site in exon 1 (∼20%) in normal individuals. All the splice variants showed steady increase in their expression from early to late stages of erythropoiesis. Our study shows that ex-vivo erythropoiesis system is an invaluable tool for studying the mechanism of globin gene regulation and switching. Identification of specific splice variants in cErCs of normal individuals and in patients with β thalassaemia could define the molecular mechanisms by which splice site mutations cause β-thalassaemia. The presence of abundant splice variants in normal controls suggests that β globin gene is regulated by multiple promoters and other regulatory elements and identification of these sequences will help in understanding the molecular basis of human globin gene regulation. Disclosures:No relevant conflicts of interest to declare.

Novel universal primer-pentaplex PCR assay based on chimeric primers for simultaneous detection of five common pig viruses associated with diarrhea

Viral pathogens associated with diarrhea in pigs include porcine circovirus 2 (PCV2), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (RVA) and C (RVC) among others. In this study, a novel universal primer-based pentaplex PCR (UP-M-PCR) assay was developed for simultaneous detection and differentiation of these five viruses. The assay uses a short-cycle multiplex amplification by chimeric primers (CP), which are virus specific, with a tail added at the 5′ end of the universal primer (UP), followed by universal amplification using UPs and a regular cycle amplification. Five universal primers with CPs (UP1-5) were designed and evaluated in an UP-based single PCR (UP–S-PCR). All five UPs were found to work efficiently and UP2 exhibited the best performance. After system optimizations, the analytical sensitivity of the UP-M-PCR, using plasmids containing the specific viral target fragments, was 5 copies/reaction for each of the five viruses irrespective of presence of a single or multiple viruses in the reaction. No cross-reaction was observed with other non-target viruses. When 273 fecal samples from clinically healthy pigs were tested, the assay sensitivity was 90.9–100%, the specificity was 98.0–100%, and the agreement rate with the UP-S-PCR was 98.5–99.6% with a Kappa value being 0.95–0.98. In summary, the UP-M-PCR developed here is a rapid and highly sensitive and specific detection method that can be used to demonstrate mixed infections in pigs with diarrhea.

Diagnostic profile of the AmplideX Fragile X Dx and Carrier Screen Kit for diagnosis and screening of fragile X syndrome and other FMR1-related disorders

ABSTRACT Introduction: In 2009, a novel, CGG repeat primed FMR1 PCR assay was designed with primers flanking the triplet repeat region, as well as a third chimeric primer complementary to the (CGG)n repeat, that was capable of amplifying alleles throughout the repeat range. This assay for the first time allowed consistent detection of large full mutation alleles with PCR, resolution of heterozygosity in females and mapping of AGG interspersions. Areas Covered: The AmplideX Fragile X Dx and Carrier Screen Kit (Asuragen, Inc.) represents a refined assay that underwent validation with sensitivity analyses for FDA approval. Single-site precision, analytical sensitivity and specificity, limit of detection and diagnostic performance were assessed in comparison to reference methods at three independent sites. Single-site precision across all genotype categories showed 100% agreement at 20 ng input across multiple operators, days, instruments and kit lots. Compared to Southern Blot analysis, the overall percent agreement was over 98% for all expanded alleles. Expert Opinion: Limitations include no methylation assessment and hard to see full mutation peaks in some mosaic samples, but overall the assay is considered a highly accurate and time-efficient assay for FMR1 allele size determination.

Specific Assay of Negative Strand Template to Quantify Intracellular Levels of Rhinovirus Double-Stranded RNA.

Human rhinovirus infections are a major trigger for acute exacerbations of lower airway diseases, including asthma and chronic obstructive pulmonary disease. Disease exacerbation is thought to be regulated via double-stranded RNA (dsRNA)-mediated signaling of proinflammatory and host defense responses in airway epithelial cells. Despite the central role of dsRNA in regulating host cell responses, no method for the quantitative assessment of dsRNA levels during HRV infections has been developed. Conventional RT-PCR for the negative strand template is not effective as self-priming results in apparent signals, even in the absence of primer during reverse transcription. To avoid these issues, we developed a selective assay for the negative strand template that uses a chimeric primer containing a 5′ non-viral sequence for reverse transcription and a primer using the non-viral sequence during subsequent PCR. We established that this assay avoided issues of self-priming and is strand specific, as it is unaffected even in the presence of a 1000-fold excess of positive strand. Assays in primary human airway epithelial cells showed that negative strand was detectable within 6 h of virus exposure and peaked at 18 h after virus exposure. The temporal pattern of negative strand induction mirrored that of genomic RNA but was always 1000-fold lower than positive strand, indicating that the negative strand levels regulate levels of dsRNA formation. This assay will permit relative quantification of dsRNA during studies of HRV regulation of epithelial cell function.

Activity and fidelity of human DNA polymerase α depend on primer structure

DNA polymerase α (Polα) plays an important role in genome replication. In a complex with primase, Polα synthesizes chimeric RNA-DNA primers necessary for replication of both chromosomal DNA strands. During RNA primer extension with deoxyribonucleotides, Polα needs to use double-stranded helical substrates having different structures. Here, we provide a detailed structure-function analysis of human Polα's interaction with dNTPs and DNA templates primed with RNA, chimeric RNA-DNA, or DNA. We report the crystal structures of two ternary complexes of the Polα catalytic domain containing dCTP, a DNA template, and either a DNA or an RNA primer. Unexpectedly, in the ternary complex with a DNA:DNA duplex and dCTP, the "fingers" subdomain of Polα is in the open conformation. Polα induces conformational changes in the DNA and hybrid duplexes to produce the universal double helix form. Pre-steady-state kinetic studies indicated for both duplex types that chemical catalysis rather than product release is the rate-limiting step. Moreover, human Polα extended DNA primers with higher efficiency but lower processivity than it did with RNA and chimeric primers. Polα has a substantial propensity to make errors during DNA synthesis, and we observed that its fidelity depends on the type of sugar at the primer 3'-end. A detailed structural comparison of Polα with other replicative DNA polymerases disclosed common features and some differences, which may reflect the specialization of each polymerase in genome replication.

Are We Ready for Fragile X Newborn Screening Testing?-Lessons Learnt from a Feasibility Study.

Fragile X syndrome (FXS) is the most prevalent heritable cause of cognitive impairment but is not yet included in a newborn screening (NBS) program within Australia. This paper aims to assess the feasibility and reliability of population screening for FXS using a pilot study in one hospital. A total of 1971 mothers consented for 2000 newborns to be tested using routine NBS dried blood spot samples. DNA was extracted and a modified PCR assay with a chimeric CGG primer was used to detect fragile X alleles in both males and females in the normal, premutation, and full mutation ranges. A routine PCR-based fragile X assay was run in parallel to validate the chimeric primer assay. Babies with CGG repeat number ≥59 were referred for family studies. One thousand nine hundred and ninety NBS samples had a CGG repeat number less than 55 (1986 < 50); 10 had premutation alleles >54 CGG repeats (1/123 females and 1/507 males). There was complete concordance between the two PCR-based assays. A recent review revealed no clinically identified cases in the cohort up to 5 years later. The cost per test was $AUD19. Fragile X status can be determined on routine NBS samples using the chimeric primer assay. However, whilst this assay may not be considered cost-effective for population screening, it could be considered as a second-tier assay to a developed immunoassay for fragile X mental retardation protein (FMRP).

A improved tetra-primer amplification refractory mutation system PCR based assay to detect four single nucleotide polymorphisms associated with folate metabolism in a single reaction

Objective To develop a tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) assay for detecting four single nucleotide polymorphisms (SNPs): rs1801133, rs1801131, rs1805087 and rs1801394 associated with folate metabolism in a single reaction tube. Methods Methodology was developed. Then, it applied the established method to analyze 150 physical examination children′s anticoagulant blood samples admitted to the Department of Clinical Laboratory, Children′s Hospital of Hebei Province from January 2017 to April 2017. Four sets of chimeric primers consisting of a universal Tag sequence and targeting rs1801133, rs1801131, rs1805087 and rs1801394 fused to the specific sequence were designed according to the T-ARMS-PCR principle and the multiplex chimeric primers strategy. A single tube PCR was then conducted after optimizing the primer concentrations and the reaction conditions. The amplified products were analyzed by QIAxcel capillary electrophoresis, and the corresponding SNP genotypes of 150 samples were identified. Furthermore, all samples were verified by direct sequencing. And the Hardy-Weinberg Equilibrium ( HWE) testing of four SNPs from 150 samples were conducted by SPSS17.0 Chi-square test. Results The improved T-ARMS-PCR combined with capillary electrophoresis can accurately verify eight different alleles of the four SNPs associated with folate metabolism at one time in 3 hours. Four SNPs of 150 whole blood samples were accurately classified and the results were completely consistent with direct sequencing. All the genotype frequencies of these four SNPs were in HWE (χrs18011332=0.69, Prs1801133=0.40; χrs18011312=0.21, Prs1801131=0.64; χrs18050872=3.32, Prs1805087=0.07; χrs18013942=1.91, Prs1801394=0.17). Conclusions The proposed improved T-ARMS-PCR combined with capillary electrophoresis in this study can accurately verify four SNPs associated with folate metabolism in a single reaction tube. This method might be a valuable tool to specifically guide the folate supplement in general population.(Chin J Lab Med, 2017, 40: 799-804) Key words: Folic acid; Metabolism; Polymorphism, single nucleotide; Polymerase Chain reaction; Electrophoresie, capillary; DNA mutational analysis; DNA primers

RT-qPCR with chimeric dU stem-loop primer is efficient for the detection of bacterial small RNAs.

Small non-coding RNAs are considered be involved in the regulation of multiple cellular processes. Quantitative reverse transcription PCR (RT-qPCR) is widely used in the detection of eukaryotic microRNA, and the stem-loop primers can improve the specificity and efficiency of reverse transcription. However, the loop structure of primers probably influence the next quantitative amplification due to the base stacking and steric hindrance. Here, we designed a chimeric stem-loop primer with a deoxyuracil (dU) base located near the RNA matching part. After the reverse transcription, uracil-DNA glycosylase (UDG) treatment was used to remove the dU base and destroy the stem-loop structure of RT product. Enzymatic assay confirmed that the recombinant UDG could efficiently eliminate the dU base in the oligonucleotide. Transcriptions of two small RNAs (TFF and ryeA) in Escherichia coli were detected by RT-qPCR with different primers. Results showed that the use of the chimeric dU stem-loop primer and UDG treatment could enhance the detection specificity and sensitivity about 1.1- to 3.4-fold, compared to those with traditional stem-loop primer and linear primer. Total RNA of 1-10pg was enough for efficient detection with the chimeric stem-loop primers. In a word, this strategy could promote the RT-qPCR detection efficiency on the transcription of bacterial small RNAs even in trace samples and can facilitate the detection of exiguous change in cellular metabolism.

Elaborated Action of the Human Primosome.

The human primosome is a 340-kilodalton complex of primase (DNA-dependent RNA polymerase) and DNA polymerase α, which initiates genome replication by synthesizing chimeric RNA-DNA primers for DNA polymerases δ and ε. Accumulated biochemical and structural data reveal the complex mechanism of concerted primer synthesis by two catalytic centers. First, primase generates an RNA primer through three steps: initiation, consisting of dinucleotide synthesis from two nucleotide triphosphates; elongation, resulting in dinucleotide extension; and termination, owing to primase inhibition by a mature 9-mer primer. Then Polα, which works equally well on DNA:RNA and DNA:DNA double helices, intramolecularly catches the template primed by a 9-mer RNA and extends the primer with dNTPs. All primosome transactions are highly coordinated by autoregulation through the alternating activation/inhibition of the catalytic centers. This coordination is mediated by the small C-terminal domain of the primase accessory subunit, which forms a tight complex with the template:primer, shuttles between the primase and DNA polymerase active sites, and determines their access to the substrate.

Development of a GeXP-multiplex PCR assay for the simultaneous detection and differentiation of six cattle viruses.

Foot-and-mouth disease virus (FMDV), Bluetongue virus (BTV), Vesicular stomatitis Virus (VSV), Bovine viral diarrheal (BVDV), Bovine rotavirus (BRV), and Bovine herpesvirus 1 (IBRV) are common cattle infectious viruses that cause a great economic loss every year in many parts of the world. A rapid and high-throughput GenomeLab Gene Expression Profiler (GeXP) analyzer-based multiplex PCR assay was developed for the simultaneous detection and differentiation of these six cattle viruses. Six pairs of chimeric primers consisting of both the gene-specific primer and a universal primer were designed and used for amplification. Then capillary electrophoresis was used to separate the fluorescent labeled PCR products according to the amplicons size. The specificity of GeXP-multiplex PCR assay was examined with samples of the single template and mixed template of six viruses. The sensitivity was evaluated using the GeXP-multiplex PCR assay on serial 10-fold dilutions of ssRNAs obtained via in vitro transcription. To further evaluate the reliability, 305 clinical samples were tested by the GeXP-multiplex PCR assay. The results showed that the corresponding virus specific fragments of genes were amplified. The detection limit of the GeXP-multiplex PCR assay was 100 copies/μL in a mixed sample of ssRNAs containing target genes of six different cattle viruses, whereas the detection limit for the Gexp-mono PCR assay for a single target gene was 10 copies/μL. In detection of viruses in 305 clinical samples, the results of GeXP were consistent with simplex real-time PCR. Analysis of positive samples by sequencing demonstrated that the GeXP-multiplex PCR assay had no false positive samples of nonspecific amplification. In conclusion, this GeXP-multiplex PCR assay is a high throughput, specific, sensitive, rapid and simple method for the detection and differentiation of six cattle viruses. It is an effective tool that can be applied for the rapid differential diagnosis of clinical samples and for epidemiological investigation.

A novel developed method based on single primer isothermal amplification for rapid detection of Alicyclobacillus acidoterrestris in apple juice

Alicyclobacillus acidoterrestris (A. acidoterrestris), as one of the most major spoilage-causing species within the Alicyclobacillus genus, can survive and multiply in the pasteurization process. Nowadays, A. acidoterrestris has become worldwide issue in the fruit juice industry due to its spore-forming and thermo-acidophilic features. A novel single primer isothermal amplification assay (SPIA) was developed for the rapid detection of A. acidoterrestris in apple juice. This assay was designed to target the 16S rRNA gene with a DNA/RNA chimeric primer. Detection of gene amplification was accomplished by amplification curve, turbidity and addition of single strand DNA binding dye SYBR Green II allowing visualization under ultra violet light. The specificity of the assay was confirmed using 7 strains of A. acidoterrestris and 30 strains of non-A. acidoterrestris. The SPIA was highly sensitive and the detection limit was as low as 4.8 CFU/mL. Furthermore, the apple juice inoculated with 61 CFU/mL of A. acidoterrestris could be detected as positive. The novel SPIA with visualization results was successfully applied for the detection of A. acidoterrestris and exhibited high specificity and sensitivity, making it a powerful tool for the detection of A. acidoterrestris in fruit juice industry and being conveniently applied in developing countries with limited resource.

Validation of a High-Throughput Multiplex Genetic Detection System for Helicobacter pylori Identification, Quantification, Virulence, and Resistance Analysis.

Helicobacter pylori (H. pylori) infection is closely related to various gastroduodenal diseases. Virulence factors and bacterial load of H. pylori are associated with clinical outcomes, and drug-resistance severely impacts the clinical efficacy of eradication treatment. Existing detection methods are low-throughput, time-consuming and labor intensive. Therefore, a rapid and high-throughput method is needed for clinical diagnosis, treatment, and monitoring for H. pylori. High-throughput Multiplex Genetic Detection System (HMGS) assay was established to simultaneously detect and analyze a set of genes for H. pylori identification, quantification, virulence, and drug resistance by optimizing the singlet-PCR and multiple primers assay. Twenty-one pairs of chimeric primers consisted of conserved and specific gene sequences of H. pylori tagged with universal sequence at the 5′ end were designed. Singlet-PCR assay and multiple primers assay were developed to optimize the HMGS. The specificity of HMGS assay was evaluated using standard H. pylori strains and bacterial controls. Six clinical isolates with known genetic background of target genes were detected to assess the accuracy of HMGS assay. Artificial mixed pathogen DNA templates were used to evaluate the ability to distinguish mixed infections using HMGS assay. Furthermore, gastric biopsy specimens with corresponding isolated strains were used to assess the capability of HMGS assay in detecting biopsy specimens directly. HMGS assay was specific for H. pylori identification. HMGS assay for H. pylori target genes detection were completely consistent with the corresponding genetic background. Mixed infection with different drug-resistant isolates of H. pylori could be distinguished by HMGS assay. HMGS assay could efficiently diagnose H. pylori infection in gastric biopsy specimens directly. HMGS assay is a rapid and high throughput method for the simultaneous identification and quantification of H. pylori, analysis of virulence and drug resistance in both isolated strains and biopsy specimens. It could also be used to distinguish the mixed infection with different resistant genotype strains. Furthermore, HMGS could detect H. pylori infection in gastric biopsy specimens directly.

Simultaneous detection of 13 viruses involved in meningoencephalitis using a newly developed multiplex PCR Mag-array system.

The early detection and identification of pathogens in central nervous system viral infections associated with neurological disease increases the survival rate. However, the limitations of current diagnostic methods contribute to a lack of proper diagnosis in 62% of patients. Therefore, a robust method for detecting multiple viruses in a single reaction with high specificity, throughput, and speed is required. A multiplex PCR Mag-Array (MPMA) system was developed that integrates three strategies: chimeric primer design, temperature switch PCR, and MagPlex-TAG techniques. The MPMA was used to amplify 13 target viral sequences simultaneously, with plasmids containing specific viral sequences as standard samples. To evaluate its clinical performance, 177 cerebrospinal fluid (CSF) samples were tested. The MPMA system presented high specificity and efficiency in detecting a control panel of 13 plasmids. Among 177 CSF samples, consistent results were achieved for 19 samples pre-tested using a commercial kit. Viral pathogens were found in 28/138 undiagnosed samples, with herpes simplex viruses (HSV-1 and HSV-2) being predominant. The 20 non-infectious samples revealed negative results. Compared to sequencing methods, sensitivity for detecting HSV-1 and HSV-2 was 100% and 98.78%, respectively, and specificity was 100% and 98.22%, respectively. A robust MPMA system that can simultaneously and reliably detect 13 meningoencephalitis-associated viruses with high specificity, throughput, and speed has been developed.

Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome

The human primosome, a 340-kilodalton complex of primase and DNA polymerase α (Polα), synthesizes chimeric RNA-DNA primers to be extended by replicative DNA polymerases δ and ϵ. The intricate mechanism of concerted primer synthesis by two catalytic centers was an enigma for over three decades. Here we report the crystal structures of two key complexes, the human primosome and the C-terminal domain of the primase large subunit (p58C) with bound DNA/RNA duplex. These structures, along with analysis of primase/polymerase activities, provide a plausible mechanism for all transactions of the primosome including initiation, elongation, accurate counting of RNA primer length, primer transfer to Polα, and concerted autoregulation of alternate activation/inhibition of the catalytic centers. Our findings reveal a central role of p58C in the coordinated actions of two catalytic domains in the primosome and ultimately could impact the design of anticancer drugs.

Simultaneous typing of seven porcine pathogens by multiplex PCR with a GeXP analyser

A novel high-throughput method was developed for simultaneous detection and differentiation of seven porcine pathogens by multiplex PCR based on a GenomeLab Gene Expression Profiler (GeXP) analyser. The pathogens included in this study were pseudorabies virus (PRV), classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), porcine circovirus type 2 (PCV-2) and Japanese encephalitis virus (JEV). Seven pairs of chimeric primers, consisting of a pathogen-specific sequence fused to a universal sequence at the 5′ end, were used to initiate the PCR, after which a set of universal primers was used for the subsequent cycles of the PCR. Amplification products were separated by capillary electrophoresis and identified using fluorescence spectrophotometry. The specificity of the GeXP assay was examined with single and mixed pathogen cDNA/DNA templates. The specific DNA product amplification peaks of seven pathogens were observed on the GeXP analyser. Negative controls did not produce DNA products. The sensitivity was evaluated by performing the assay on serial 10-fold dilutions of the plasmids containing the target sequence. Under optimised conditions this assay achieved a sensitivity of 100–1000 copies/μL for a single virus and 1000 copies/μL when all of the seven pre-mixed viral targets were present. Furthermore, the GeXP-PCR assay was 100% specific when 58 clinical samples were tested in comparison with the conventional PCR method. In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for simultaneously detecting seven pathogens that infect swine.

Molecular characterization of X chromosome fragility in idiopathic mental retardation

BackgroundFragile X syndrome (FXS) is the most common form of inherited mental retardation. Frequency of fragile X syndrome among male siblings and relatives of mentally retarded patients is relatively high. Cytogenetic diagnosis of FXS is unreliable since it is ineffective for the diagnosis of premutated males or females. Proper molecular diagnosis is a pre-requisite for providing proper counseling advice. Subjects and methodsSixty-four males with idiopathic mental retardation, ranging in age from 4.2 to 19years (10.92±4.00) were clinically pre-selected, based on scoring protocol comprising eight features of the syndrome, before molecular testing. A rapid polymerase chain reaction-based screening was applied for detection of expanded FMR1 alleles. Samples that did not yield the normal band lengths were subjected to a second PCR screen. The secondary screen utilizes a chimeric primer demonstrating the presence or absence of an expanded allele. ResultsAmplification of FMRI gene by PCR of tested patients revealed that 8 cases (12.5%) have full mutation and 6 cases (9.4%) have premutation. A wide range of Fra X-scoring ranging from 1 to 7 features was detected in examined cases. Significant clinical features included large prominent ears, hyperextensibility of joints and macroorchidism in post pubertal males. ConclusionsA simplified checklist of fragile X should be used for patients with idiopathic MR and those patients above score 3 should be tested for FXS. The diagnostic assay may be used as a screening method for fragile X syndrome being rapid and cost effective compared to other techniques. In addition, screening of all relatives of proven patients should be performed to detect clinically unidentified cases for provision of proper counseling and optimal management of detected cases.

Simultaneous detection of eight swine reproductive and respiratory pathogens using a novel GeXP analyser-based multiplex PCR assay

A new high-throughput GenomeLab Gene Expression Profiler (GeXP) analyser-based multiplex PCR assay was developed for the detection of eight reproductive and respiratory pathogens in swine. The reproductive and respiratory pathogens include North American porcine reproductive and respiratory syndrome virus (PRRSV-NA), classical swine fever virus (CSFV), porcine circovirus 2 (PCV-2), swine influenza virus (SIV) (including H1 and H3 subtypes), porcine parvovirus (PPV), pseudorabies virus (PRV) and Japanese encephalitis virus (JEV). Nine pairs of specific chimeric primers were designed and used to initiate PCRs, and one pair of universal primers was used for subsequent PCR cycles. The specificity of the GeXP assay was examined using positive controls for each virus. The sensitivity was evaluated using serial ten-fold dilutions of in vitro-transcribed RNA from all of the RNA viruses and plasmids from DNA viruses. The GeXP assay was further evaluated using 114 clinical specimens and was compared with real-time PCR/single RT-PCR methods. The specificity of the GeXP assay for each pathogen was examined using single cDNA/DNA template. Specific amplification peaks of the reproductive and respiratory pathogens were observed on the GeXP analyser. The minimum copies per reaction detected for each virus by the GeXP assay were as follows: 1000 copies/μl for PRV; 100 copies/μl for CSFV, JEV, PCV-2 and PPV; and 10 copies/μl for SIV-H1, SIV-H3 and PRRSV-NA. Analysis of 114 clinical samples using the GeXP assay demonstrated that the GeXP assay had comparable detection to real-time PCR/single RT-PCR. This study demonstrated that the GeXP assay is a new method with high sensitivity and specificity for the identification of these swine reproductive and respiratory pathogens. The GeXP assay may be adopted for molecular epidemiological surveys of these reproductive and respiratory pathogens in swine populations.