Multiplex Research Articles

A SMART method for isolating monoclonal antibodies from individual rhesus macaque memory B cells

Characterizing antigen-specific B cells is a critical component of vaccine and infectious disease studies in rhesus macaques (RMs). However, it is challenging to capture immunoglobulin variable (IgV) genes from individual RM B cells using 5′ multiplex (MTPX) primers in nested PCR reactions. In particular, the diversity within RM IgV gene leader sequences necessitates large 5′ MTPX primer sets to amplify IgV genes, decreasing PCR efficiency. To address this problem, we developed a switching mechanism at the 5′ ends of the RNA transcript (SMART)-based method for amplifying IgV genes from single RM B cells to capture Ig heavy and light chain pairs. We demonstrate this technique by isolating simian immunodeficiency virus (SIV) envelope-specific antibodies from single-sorted RM memory B cells. This approach has several advantages over existing methods for cloning antibodies from RMs. First, optimized PCR conditions and SMART 5′ and 3′ rapid amplification of cDNA ends (RACE) reactions generate full-length cDNAs from individual B cells. Second, it appends synthetic primer binding sites to the 5′ and 3′ ends of cDNA during synthesis, allowing for PCR amplification of low-abundance antibody templates. Third, the nested PCR primer mixes are simplified by employing universal 5′ primers, eliminating the need for complex 5′ MTPX primer sets. We anticipate this method will enhance the isolation of antibodies from individual RM B cells, supporting the genetic and functional characterization of antigen-specific B cells.

A novel 5x multiplex immunohistochemical staining reveals PSMA as a helpful marker in prostate cancer with low p504s expression.

The ability to combine multiple immunohistochemical (IHC) markers within a single tissue section facilitates the evaluation and detection of co-expressions, while saving tissue. A newly developed 5x multiplex (MPX) IHC staining of five different IHC markers (Basal cell cocktail (34βE12 + p63), p504s (SP116), ERG (EPR3864), Ki-67 (30−9), PSMA (EP192)) was applied on whole sections of n = 37 radical prostatectomies (RPE) including normal and cancerous tissue. Four different colors including brown, magenta, yellow and teal coded for different stainings, whereas magenta was used twice for nuclear Ki-67 and cytosolic / membranous PSMA. The staining of multiplex IHC was compared to single stains of ERG, PSMA and p504s. The proper staining of the basal cell cocktail and Ki-67 could be assessed by internal positive controls in the multiplex staining. The proportion of PSMA and p504s expression revealed a significant correlation between multiplex and single stains (p < 0.01) as well as a concordant staining pattern for ERG (n = 14 prostate cancers were identified ERG positive with both methods). Our proof of concept study demonstrates a robust staining pattern of all five different antibodies with this newly developed 5x MPX IHC. This approach facilitates the recognition of prostate cancer, in particular by adding PSMA in cases with low p504s expression.

Design and Analysis of 32 bit R evers e C onverter based on low power Parallel Prefix A dder

The Residue Number System (RNS) based reverse converter can play as main role in Parallel arithmetic operations of Digital Signal Processing (DSP) applications and VLSI technologies. Normally, by the use of carry adders, the reverse conversion design gives high delay and high power consumption. Due to resolve of above problem, the design of reverse converter is proposed by the use of familiar high speed (less propagation delay) Parallel Prefix - Kogge Stone Adder (PP- KSA). This paper describes the design of 32-bit Reverse converter with regular PP-KSA and proposed MUX (Multiplex) logic of PP-KSA with Hybrid Modular Parallel Prefix structure (HMPE) separately. In addition to that, the performance of that designs are analysed based on area, delay and power independently. The Performance results of proposed MUX logic of PP-KSA Reverse converter design yields low power than the other design which uses the regular PP-KSA. The simulation and synthesis effects can be done in Xilinx ISE 14.2i tool.

Differences in Cognition and Behaviour in Multiplex and Simplex Autism: Does Prior Experience Raising a Child with Autism Matter?

Previous research has found multiplex (MPX) children have an advantage in cognition compared to simplex (SPX) children. However, MPX parent's previous experience with older diagnosed siblings has not been considered. We used a large database sample to investigate the MPX advantage and contribution of birth order. Children from the Autism Genetic Resource Exchange (AGRE) were stratified into first- (MPX1, n = 152) and second-affected MPX (MPX2, n = 143), SPX (n = 111), and only-child SPX (SPXOC, n = 23) groups. Both MPX groups had higher cognitive scores compared to SPX groups, with no differences between MPX1 and MPX2 groups. No differences were found for autism symptoms or adaptive behaviour. These results suggest parent experience due to birth order is an unlikely contributor to the MPX cognitive advantage.

Cognitive and behavioral differences in toddlers with autism spectrum disorder from multiplex and simplex families.

Prospective, longitudinal designs utilizing "high-risk" infant siblings of children diagnosed with Autism Spectrum Disorder (ASD-sibs) have provided unique and valuable insights regarding the early ASD phenotype. However, it remains unclear whether these cases are representative of all children with ASD. The objective in the present study was to investigate whether the early development of toddlers with ASD from multiplex (MPX) families, who have an affected older sibling, is similar or different to toddlers with ASD from simplex (SPX) families, where there is no affected sibling. A further aim was to examine patterns of association between autism symptom severity and cognitive functioning within each group to inform possible mechanisms for group similarities/differences. Behavioral and cognitive assessment data from a sample of toddlers with ASD was utilized, comprising 45 MPX, 127 first-born SPX, and 72 later-born SPX toddlers. Participants in the MPX group had significantly higher developmental quotients on the Mullen Scales of Early Learning compared to those in the SPX groups, who did not differ from each other. However, all three groups were similar on their autism severity scores (measured using the Autism Diagnostic Observation Schedule and the Autism Diagnostic Interview), and the pattern of relationships between cognitive ability and autism symptom severity. The results suggest that caution be exercised in generalizing findings from ASD-sib samples to other samples of children with ASD. The higher cognitive abilities in the MPX group, in addition to biological differences, may also be an outcome of family environmental factors, which deserves further investigation. Autism Research 2019, 12: 682-693. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: We sought to establish whether toddlers with autism from families where there is more than one affected child, called multiplex families, are different to children from simplex families, where there is only one affected child, and no other members within the immediate family with an autism diagnosis. We found that while toddlers from multiplex families were similar to those from simplex families in their autism symptoms, they were more developmentally advanced than children in the latter group.

MP67-10 IDENTIFICATION OF URINARY MACROPHAGE-RELATED FACTORS SPECIFIC TO PATIENTS WITH CALCIUM OXALATE STONES USING MULTIPLEX ANALYSIS

You have accessJournal of UrologyStone Disease: Basic Research & Pathophysiology II1 Apr 2016MP67-10 IDENTIFICATION OF URINARY MACROPHAGE-RELATED FACTORS SPECIFIC TO PATIENTS WITH CALCIUM OXALATE STONES USING MULTIPLEX ANALYSIS Atsushi Okada, Kazumi Taguchi, Yasuhiro Fujii, Shuzo Hamamoto, Masahito Hirose, Yasunori Itoh, Keiichi Tozawa, Shoichi Sasaki, Yutaro Hayashi, Kenjiro Kohri, and Takahiro Yasui Atsushi OkadaAtsushi Okada More articles by this author , Kazumi TaguchiKazumi Taguchi More articles by this author , Yasuhiro FujiiYasuhiro Fujii More articles by this author , Shuzo HamamotoShuzo Hamamoto More articles by this author , Masahito HiroseMasahito Hirose More articles by this author , Yasunori ItohYasunori Itoh More articles by this author , Keiichi TozawaKeiichi Tozawa More articles by this author , Shoichi SasakiShoichi Sasaki More articles by this author , Yutaro HayashiYutaro Hayashi More articles by this author , Kenjiro KohriKenjiro Kohri More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1326AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We previously demonstrated the involvement of inflammatory macrophages (M1-Mϕ) and anti-inflammatory macrophages (M2-Mϕ) in the calcium oxalate (CaOx) kidney stone formation process by basic studies using an animal model. However, in clinical medicine, the correlations between stone formation risk and Mϕ activity have not been examined. The aim of the present study was to analyze the characteristics of CaOx stone formation factors and detect the urinary Mϕ-related factors specific to stone formation. METHODS Subjects were 249 men aged 20?79 years old who visited Nagoya City University Hospital. Of them, 111 had no history of stone formation (non-stone formers [NSF]), while 130 had a history of CaOx stone formation (stone formers [SF]): 41 had experienced one episode of stone formation and 89 had a history of recurrent stone formation. All patients had normal urinalysis results and denied a history of malignancy, collagen vascular disease, or the use of immunosuppressive or steroid medications. Using 25 µL of spot urine, we determined the concentration of 18 urinary candidate proteins using multiplex (MP) analysis on a MagPix® system. For the factors that demonstrated significant differences between NSF and SF, we performed multi- and univariate analyses for patient age, body mass index, blood and urine biochemistry data, and MP analysis data and calculated the odds ratio and 95% confidence interval of the CaOx SF using logistic regression analysis. For each parameter, the median was used as the cut-off value. RESULTS We found no significant differences in the background data or urine biochemistry between NSF and SF. The two-stage MP analyses demonstrated eight factors with significant differences between the two groups. Logistic regression analyses revealed that lower values of interleukin (IL)-4 (≤4.4 pg/mL), IL-1a (≤7.9 pg/mL), and granulocyte colony-stimulating factor (≤14.5 pg/mL) were statistically significant independent factors predictive of stone formation. Furthermore, on the validation analyses, we found significantly lower values than NSF group regardless of first time or recurrence. CONCLUSIONS CaOx SF show a significantly lower M2-Mϕ activity-related urinary IL-4 level than NSF. In CaOx SF, decreased function of M2-Mϕ, including phagocytosis and processing of CaOx crystals, may play a more important role in the stone formation process than urinary biochemical abnormalities. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e884 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Atsushi Okada More articles by this author Kazumi Taguchi More articles by this author Yasuhiro Fujii More articles by this author Shuzo Hamamoto More articles by this author Masahito Hirose More articles by this author Yasunori Itoh More articles by this author Keiichi Tozawa More articles by this author Shoichi Sasaki More articles by this author Yutaro Hayashi More articles by this author Kenjiro Kohri More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Tetracyclines downregulate the production of LPS-induced cytokines and chemokines in THP-1 cells via ERK, p38, and nuclear factor-κB signaling pathways

Recent reports have shown that antibiotics such as macrolide, aminoglycoside, and tetracyclines have immunomodulatory effects in addition to essential antibiotic effects. These agents may have important effects on the regulation of cytokine and chemokine production. However, the precise mechanism is unknown. This time, we used Multi Plex to measure the production of cytokines and chemokines following tetracycline treatment of lipopolysaccharide (LPS)-induced THP-1 cells. The signaling pathways were investigated with Western blotting analysis. Three tetracyclines significantly suppressed the expression of cytokines and chemokines induced by LPS. Minocycline (50μg/ml), tigecycline (50μg/ml), or doxycycline (50μg/ml) were added after treatment with LPS (10μg/ml). Tumor necrosis factor-α was downregulated to 16%, 14%, and 8%, respectively, after 60min compared to treatment with LPS without agents. Interleukin-8 was downregulated to 43%, 32%, and 26% at 60min. Macrophage inflammatory protein (MIP)-1α was downregulated to 23%, 33%, and 16% at 120min. MIP-1β was downregulated to 21%, 11%, and 2% at 120min. Concerning about signaling pathways, the mechanisms of the three tetracyclines might not be the same. Although the three tetracyclines showed some differences in the time course, tetracyclines modulated phosphorylation of the NF-κB pathway, p38 and ERK1/2/MAPK pathways, resulting in inhibition of cytokine and chemokine production. In addition, SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly suppressed the expression of TNF-α and IL-8 in LPS-stimulated THP-1 cells. And further, the NF-κB inhibitor, BAY11-7082, almost completely suppressed LPS-induced these two cytokines production. Thus, more than one signaling pathway may be involved in tetracyclines downregulation of the expression of LPS-induced cytokines and chemokines in THP-1 cells. And among the three signaling pathways, NF-κB pathway might be the dominant pathway on tetracyclines modification the LPS-induced cytokines production in THP-1 cells. In general, minocycline and doxycycline suppressed the production of cytokines and chemokines in LPS-stimulated THP-1 cell lines via mainly the inhibition of phosphorylation of NF-κB pathways. Tigecycline has the same structure as the other tetracyclines, however, it showed the different properties of cytokine modulation in the experimental time course.

Cognitive impairments are different in single-incidence and multi-incidence ADHD families.

We may improve our understanding of the role of common versus unique risk factors in attention-deficit/hyperactivity disorder (ADHD) by examining ADHD-related cognitive deficits in single- (SPX), and multi-incidence (MPX) families. Given that individuals from multiplex (MPX) families are likely to share genetic vulnerability for the disorder, whereas simplex (SPX) ADHD may be the result of sporadic (non-)genetic causes unique to the patient, we hypothesized that cognitive impairments may be different in SPX and MPX ADHD as indicated by (a) the presence of cognitive deficits in MPX, but not SPX unaffected siblings and (b) dissimilar cognitive profiles in SPX and MPX ADHD patients. Tasks measuring total IQ, verbal attention, executive functioning, motor functioning, and time estimation were administered to 31 SPX/264 MPX ADHD probands, 47 SPX/123 MPX unaffected siblings, and 263 controls, aged 6-19 years. SPX unaffected siblings were unimpaired compared to controls, except for verbal working memory, whereas MPX unaffected siblings showed impairments on most cognitive domains. The cognitive profiles of SPX and MPX probands were highly similar, except that verbal attention, response inhibition and motor control deficits were more pronounced in MPX probands, and -compared to their unaffected siblings- impairments in IQ, visual working memory and timing abilities were more pronounced in SPX cases. Our results support the hypothesis that a partly different cognitive architecture may underlie SPX and MPX forms of ADHD, which becomes evident when contrasting cognitive performances within families. Cognitive factors underlying MPX forms of ADHD are familial, whereas nonfamilial in SPX ADHD. SPX-MPX stratification may be a step forward in unraveling diverse causal pathways.

Multiplex PCR assay for the detection of common dermatophyte nail infections

Onychomycosis is one of the most prevalent dermatophytic diseases. Mycological methods used in the conventional diagnosis may not be optimal. Multiplex (MX) PCR was reported as a reliable alternative. Dermatophyte gene sequence records were used to design a MX PCR for detection and identification of dermatophytes in nail specimens. A MX PCR method based on the amplification of the chitin synthase 1 and internal transcribed spacer genes was developed. The study included 93 strains of dermatophytes and non-dermatophytic fungi, six dermatophytic reference strains and 201 nail specimens from patients with dermatophytic onyxis. DNA extraction directly from nail samples was carried out by using the QIAamp DNA extraction kit (Quiagen). A set of primers was designed and their specificity was assessed. MX PCR detected the causal agent in specimens from which Trichophyton rubrum and T. interdigitale grew in culture and also identified a dermatophyte species in an additional 32 specimens that were negative in microscopy and culture. None of the investigated non-dermatophytic strains was positive. Sensitivity of MX PCR was higher as compared to mycological examination (97% vs. 81.1%). MX PCR for direct detection of dermatophytes from nail samples yielded mixed flora in 32.8% of samples. MX PCR proved sensitive and adequate for the diagnosis of dermatophytic onychomycosis. It is much adapted to cases where culture is negative or contaminated by overgrowing moulds, which makes the identification of the causal agent problematic.

Evidence for broader autism phenotype characteristics in parents from multiple‐incidence autism families

The broader autism phenotype (BAP) was assessed in parents who have two or more children with autism spectrum disorder (ASD) (multiplex (MPX) autism), parents who have no more than one child with ASD (simplex autism), parents who have a child with developmental delay without ASD, and parents who have typically developing children. Clinicians, naive to parent group membership status, rated BAP characteristics from videotaped administration of the Broader Autism Phenotype Symptom Scale (BPASS). Differences among groups in BPASS scores in the four assessed domains (social motivation, conversational skills, expressiveness, and restricted interests) were examined using multivariate ANOVA and post hoc comparisons. Further, ratings of videotapes by observers naïve to family status were compared with live, non-naive ratings by observers who were aware of family status (non-naïve). Findings demonstrate that the BPASS is an instrument resistant to rater bias. Parents from MPX autism families showed significantly more autism phenotype characteristics than the parents in the other groups. Moreover, the parents from simplex autism families did not differ from the parents of children with developmental delay or typical development. Finally, no differences between live, non-naive ratings and videotaped, naive ratings were observed. These findings suggest that characteristics of the BAP, specifically in the social and communication domains, are present in MPX autism parents to a greater degree than simplex autism and control parents. Further, the results provide support for the notion that genetic transmission mechanisms may differ between families with more than one child with autism and families with only one child with autism.

Sensitivity comparison of two Food and Drug Administration–licensed, triplex nucleic acid test automated assays for hepatitis B virus DNA detection and associated projections of United States yield

There have been no comparisons of the relative sensitivity of the two Food and Drug Administration-licensed multiplex (MPX) nucleic acid test (NAT) systems (Procleix Ultrio [Gen-Probe], TIGRIS platform [Novartis]; and cobas TaqScreen MPX [Roche Molecular Systems], cobas s 201 platform [Roche Instrument Center]) for detecting hepatitis B virus (HBV)-infected donors in minipool sizes (MP) used in the United States. Routine blood samples from Thailand were obtained from plasma units from 129 hepatitis B surface antigen (HBsAg)-negative, HBV NAT-yield donations. Blinded US testing included antibody to hepatitis B core antigen (anti-HBc), NAT using both manufacturers' systems (undiluted-individual donation [ID], in singlet and diluted 1:6 and 1:16 in triplicate), quantitative antibody to hepatitis B surface antigen, HBV DNA viral loads, and HBV genotyping. HBV yields in the United States were estimated using the incidence/window period (WP) model and compared to the calculated assay sensitivities. Eighty samples were classified as occult HBV (anti-HBc reactive) and 49 as WP (anti-HBc nonreactive). For US pool sizes, MPX detected significantly more samples than Ultrio (MPX MP6 vs. Ultrio MP16; p < 0.0001 for occult and WP). Ultrio MP16 results were not statistically different from Ultrio MP6 (p = 0.68 for occult; p = 0.42 for WP). There was no difference between platforms for MP sizes used in most of the world (MPX MP6 vs. Ultrio ID; p = 0.70 for occult and p = 0.34 for WP). Viral loads were higher in WP samples. Modeled yield estimates were consistent with measured assay sensitivity on the Thai donor samples. As used in the United States, MPX MP6 is more sensitive than Ultrio MP16, but the impact of this difference is mitigated by low numbers of HBV WP infections.

Modification of the BAX® System PCR Assay for Detecting Salmonella in Beef, Produce, and Soy Protein Isolate

The BAX System PCR assay for Salmonella detection in foods was previously validated as AOAC Research Institute (RI) Performance Tested Method (PTM) 100201. New studies were conducted on beef and produce using the same media and protocol currently approved for the BAX System PCR assay for E. coli O157:H7 multiplex (MP). Additionally, soy protein isolate was tested for matrix extension using the U.S. Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM) enrichment protocols. The studies compared the BAX System method to the U.S. Department of Agriculture culture method for detecting Salmonella in beef and the FDA-BAM culture method for detecting Salmonella in produce and soy protein isolate. Method comparison studies on low-level inoculates showed that the BAX System assay for Salmonella performed as well as or better than the reference method for detecting Salmonella in beef and produce in 8-24 h enrichment when the BAX System E. coli O157:H7 MP media was used, and soy protein isolate in 20 h enrichment with lactose broth followed by 3 h regrowth in brain heart infusion broth. An inclusivity panel of 104 Salmonella strains with diverse serotypes was tested by the BAX System using the proprietary BAX System media and returned all positive results. Ruggedness factors involved in the enrichment phase were also evaluated by testing outside the specified parameters, and none of the factors examined affected the performance of the assay.

Population- and Family-Based Studies Associate the MTHFR Gene with Idiopathic Autism in Simplex Families

Two methylenetetrahydrofolate reductase gene (MTHFR) functional polymorphisms were studied in 205 North American simplex (SPX) and 307 multiplex (MPX) families having one or more children with an autism spectrum disorder. Case-control comparisons revealed a significantly higher frequency of the low-activity 677T allele, higher prevalence of the 677TT genotype and higher frequencies of the 677T-1298A haplotype and double homozygous 677TT/1298AA genotype in affected individuals relative to controls. Family-based association testing demonstrated significant preferential transmission of the 677T and 1298A alleles and the 677T-1298A haplotype to affected offspring. The results were not replicated in MPX families. The results associate the MTHFR gene with autism in SPX families only, suggesting that reduced MTHFR activity is a risk factor for autism in these families.

Application of a multiplex PCR to cervical cells collected by a paper smear for the simultaneous detection of all mucosal human papillomaviruses (HPVs) and typing of high-risk HPV types 16 and 18

A simple paper smear (PS) method for dry collection and storage of cervical specimens was employed to develop an easy multiplex (MPX) PCR for simultaneous detection of generic human papillomaviruses (HPVs) as well as typing of the high-risk HPV-16 and -18, the two clinically most important HPV genotypes, which are responsible for more than 80 % of cervical cancers. Multiplexing was performed with a small amount of DNA eluted by boiling from a single PS punch in a single tube and using a mixture of four pairs of primers specific for the HPV L1 consensus sequence, HPV-16, HPV-18 and the β-globin gene. Sixty HPV-positive biopsies and corresponding PS specimens from cervical cancer patients as well as cervical smears from 100 healthy women with or without abnormal cytology were collected both as PSs and in PBS. Detection of HPV DNA from cervical biopsies collected in PBS and corresponding cervical scrapes on a PS or in PBS by conventional and MPX-PCR showed a concordance of 100 % and adequacy of 93 %. A similar comparative study in cervical scrapes from normal women also revealed 100 % concordance. The technique was validated in a multicentric study at four different national laboratories. PSs collected by different centres showed variable adequacy (73-82 %) but the use of multiple PS discs for DNA extraction significantly increased the adequacy. Integration of PSs with MPX-PCR for the detection and typing of HPVs is a highly convenient, efficient, simple and cost-effective method for large-scale clinico-epidemiological studies and is also suitable for HPV vaccine monitoring programmes in resource-poor settings.

One‐year experience of nucleic acid technology testing for human immunodeficiency virus Type 1, hepatitis C virus, and hepatitis B virus in Thai blood donations

Blood donations collected at the National Blood Center, the Thai Red Cross Society, Bangkok, in 2007 were tested by nucleic acid amplification technology (NAT) using the Chiron TIGRIS/Procleix Ultrio test and the Roche cobas s 201/cobas TaqScreen multiplex (MPX) test. The sensitivity, specificity, and robustness were determined by testing 486,676 seronegative blood donations. Samples from each day of collection were divided into two sets; the odd-numbered samples were tested individually on the TIGRIS and the even-numbered samples were tested in pools of 6 on the cobas s 201. The status of reactive samples was confirmed by duplicate testing of samples from the plasma bag to calculate the test specificity. Reactive samples were tested on the alternate system and followed up. The analytical sensitivity of both systems met the 95% limits of detection claimed by the respective package inserts. No cross contamination was seen with either system. Test specificity was 99.93 and 99.90% for the Procleix Ultrio and cobas TaqScreen tests, respectively. The NAT yield rates for human immunodeficiency virus Type 1 (HIV-1), hepatitis C virus (HCV), and hepatitis B virus (HBV) were 1:97,000, 1:490,000, and 1:2800, respectively. Several occult HBV donors, the majority of whom were detected by both tests, were also identified. The HIV-1 and HCV window cases were detected with both tests. The performances of the systems and tests indicated that both were acceptable for routine NAT by the National Blood Center, the Thai Red Cross Society. However, the Procleix Ultrio test appeared to be less sensitive than the cobas TaqScreen test for HBV.

Development and test of 21 multiplex PCRs composed of SSRs spanning most of the apple genome

A series of 21 multiplex (MP) polymerase chain reactions containing simple sequence repeat (SSR) markers spanning most of the apple genome has been developed. Eighty-eight SSR markers, well distributed over all 17 linkage groups (LGs), have been selected. Eighty-four of them were included in 21 different MPs while four could not be included in any MPs. The 21 MPs were then used to genotype approximately 2,000 DNA samples from the European High-quality Disease-Resistant Apples for a Sustainable agriculture project. Two SSRs (CH01d03 and NZAL08) were discarded at an early stage as they did not produce stable amplifications in the MPs, while the scoring of the multilocus (ML) SSR Hi07d11 and CN44794 was too complex for large-scale genotyping. The testing of the remaining 80 SSRs over a large number of different genotypes allowed: (1) a better estimation of their level of polymorphism; as well as of (2) the size range of the alleles amplified; (3) the identification of additional unmapped loci of some ML SSRs; (4) the development of methods to assign alleles to the different loci of ML SSRs and (5) conditions at which an SSR previously described as ML would amplify alleles of a single locus to be determined. These data resulted in the selection of 75 SSRs out of the 80 that are well suited and recommended for large genotyping projects.

The DLX1and DLX2 genes and susceptibility to autism spectrum disorders

An imbalance between excitation and inhibition in the cerebral cortex has been suggested as a possible etiology of autism. The DLX genes encode homeodomain-containing transcription factors controlling the generation of GABAergic cortical interneurons. The DLX1 and DLX2 genes lie head-to-head in 2q32, a region associated with autism susceptibility. We investigated 6 Tag SNPs within the DLX1/2 genes in two cohorts of multiplex (MPX) and one of simplex (SPX) families for association with autism. Family-based association tests showed strong association with five of the SNPs. The common alleles of rs743605 and rs4519482 were significantly associated with autism (P<0.012) in the first sample of 138 MPX families, with the latter remaining significant after correction for multiple testing (P(cor)=0.0046). Findings in a second sample of 169 MPX families not only confirmed the association at rs4519482 (P=0.034) but also showed strong allelic association of the common alleles at rs788172, rs788173 and rs813720 (P(cor)=0.0003-0.04). In the combined MPX families, the common alleles were all significantly associated with autism (P(cor)=0.0005-0.016). The GGGTG haplotype was over transmitted in the two MPX cohorts and the combined samples [P(cor)<0.05: P(cor)=0.00007 for the combined MPX families, Odds Ratio: 1.75 (95% CI: 1.33-2.30)]. Further testing in 306 SPX families replicated the association at rs4519482 (P=0.033) and the over transmission of the haplotype GGGTG (P=0.012) although P-values were not significant after correction for multiple testing. The findings support the presence of two functional polymorphisms, one in or near each of the DLX genes that increase susceptibility to, or cause, autism in MPX families where there is a greater genetic component for these conditions.

Detection of Infectious Bursal Disease Virus in Various Lymphoid Tissues of Experimentally Infected Specific Pathogen Free Chickens by Different Reverse Transcription Polymerase Chain Reaction Assays

Infectious bursal disease (IBD) is a worldwide distributed immunosuppressive viral disease in young chickens, controlled by vaccination. Emergence of several strains of IBD virus (IBDV) has created a demand for strain-specific diagnostic tools. In the present experiment, five different reverse transcription polymerase chain reaction (RT-PCR) assays, including two recently developed strain-specific assays, were employed for detection of ribonucleic acid (RNA) from three different IBDV strains in bursa tissue samples from experimentally infected specific pathogen free chickens. The virus strains included vaccine strain D78, classical strain Faragher 52/70, and the very virulent Danish strain DK01. The presence of the virus infection was confirmed by histopathologic evaluation of bursa lesions. The largest number of positive samples was obtained with a strain-specific two-step multiplex (MPX) RT-PCR assay based on iScript enzyme, and the commercially available Qiagen one-step RT-PCR. Between these methods, agreement was obtained for 57 of 59 samples. Because the Qiagen one-step RT-PCR assay was suggested as the more sensitive of these two assays, it was used for detection of IBDV in bone marrow, spleen, thymus, and cecal tonsils from experimentally infected chickens. The identity of the virus strains involved was confirmed by MPX RT-PCR. In conclusion, the MPX RT-PCR represented a reliable assay for detection and differentiation of IBDV strains in selected lymphoid tissues of chickens. All three of the IBDV strains used were detected in bursa tissues, whereas only the two virulent strains were detected in bone marrow, spleen, and thymus.

Multiplex PCR for the assignment of some major branches of the Y chromosome tree.

In this paper we describe a multiplex PCR useful to as-sign some major Y chromosome tree branches (DE, G2, I, J and J2) (for the nomenclature of the Y chromosome tree branches, see YCC 2002). These are prev alent in all Southern European populations, accoun ting between 51% and 77% of the total male population (Di Giacomo et al. 2003 and personal data). The identification of the allelic state at every locus in the study is done either directly (presence/a bsenc e of the specific PCR fragment), or can be simply obtained with further sequential steps of dot-blot hybrid ization with specific probes. In the last years a large number of single nucleotide polymorphic (SNP) binary markers have been identified in the non-recombining portion of the human Y chrom o-some (NRY) (YCC 2002). SNPs are ideal genetic mar k-ers for med ical and population genetic studie s, since they are widespread in the human genome and are easily de-tected by PCR based methods. In general, the SNPs ex-hibit only two, evolutionarily stable, alleles. This ad van t-age is often counterbalanced by the necessity of typing large numbers of them in many individuals. Genotyping for these markers is of great importance in forensics and in association studies. Moreover, since the NRY lacks genetic recombination during meioses, it is a pow erful tool in phylogenetic studies. In order to increase the pro-ductivity of such studies, the first choice will be to study simultaneously more SNPs in a single multiplex PCR reaction. Two papers concerning the use of mult iplex PCR in the study of the human Y chromosome SNP variation have been published recently. One of them uses a mini-sequencing/capillary electrophoresis method for Y chro-mosome typing in forensic studies (Sanchez et al. 2003); the other one uses a primer extension/mass spectroscopy method for high throughput SNP genotyping of the Y chromosome (Paracchini et al. 2002). The method pre-sented here provides a reliable effort-effective, time-effec -tive and cost-effective method for the early stages of human Y chromosome genotyping, being particularly useful when little information or no information at all is available about the genetic structure of the population under study.

Hepatitis B NAT virus‐positive blood donors in the early and late stages of HBV infection: analyses of the window period and kinetics of HBV DNA

The Japanese Red Cross (JRC) carries out nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1) by using a multiplex (MPX) reagent. Screening is undertaken on serologically negative units. In this study we characterized HBV NAT-positive donations individually and analysed the window period and kinetics of HBV DNA, during acute infection, in follow-up studies. Two hundred and seventy-seven HBV DNA-positive donations have been identified in Japan since the introduction of NAT screening of 50-donation minipools. The viral loads and genotypes of these HBV DNA-positive donations were characterized. The doubling time and half-life of HBV was estimated from the data of 123 follow-up donors. The sensitivity of the NAT system (based on 50-donation minipools) was compared with the sensitivities of the enzyme immunoassay (EIA) and the chemiluminescence immunoassay (CLIA). Samples that were CLIA negative, but with > 10(4) copies/ml of HBV DNA, were analysed by sequencing the hepatitis B surface antigen (HBsAg) region. Out of 277 HBV NAT-positive samples, 125 (45%) were found to have an increasing viral load and 45 (16%) a decreasing viral load. Forty per cent of HBV NAT-positive samples with an increasing viral load, and 33% of those with a decreasing viral load, were negative when tested by using the CLIA. No mutations related to escape mutants were found in the samples that were CLIA negative but with HBV DNA loads of > 10(4) copies/ml. The median HBV doubling time was 2.6 days (n = 93, 1.3-15.2 days) and the half-life was 1.6 days (n = 55, 0.9-6.3 days). Some kinetic difference was observed between genotypes A and B. HBV NAT screening detected HBV DNA in both early (the so-called serological window period) and late stages of acute HBV infection.