This study shows that the addition of a consensus 4-locus set of hypervariable mycobacterial interspersed repetitive-unit-variable-number tandem repeat (MIRU-VNTR) loci to the spoligotyping-24-locus MIRU-VNTR typing strategy is a well-standardized approach that can contribute to an improvement of the true cluster definition while retaining high typeability in non-Beijing strains.

Factor XIII (FXIII) deficiency is an extremely rare hemorrhagic disorder with an approximate worldwide incidence of one per two million. With current tests, diagnosis of this disease can be made more precisely. However, factors such as the number of patients with FXIII deficiency (FXIIID), available diagnostic coagulation tests and the number of molecular studies have affected the diagnosis of FXIIID in different parts of the world. Various laboratory approaches can be used, including screening and diagnosis of the disorder in countries with a relatively high rate of FXIIID and recurrent mutation(s) with a simple polymerase chain reaction-restriction fragment length polymorphism analysis or polymerase chain reaction-sequencing for detection of one or a few specific mutations. In other countries, two different laboratory approaches can be used, depending on available coagulation tests. In less-equipped coagulation laboratories, the clot solubility test remains the only diagnostic test for FXIIID. Even in these countries, at least one referral laboratory should perform FXIII activity and, if possible, confirmation of FXIIID by molecular analysis. In countries with well equipped coagulation laboratories, FXIII activity should be used to screen suspected FXIIID patients; more specific tests such as molecular analysis should be used for confirmation. This study suggests a simple, reliable and flexible algorithm for early diagnosis of FXIIID, and may, with one-time diagnosis of FXIIID, reduce the rate of morbidity and mortality in patients with the disorder.

Infra-species taxonomy is a prerequisite to compare features such as virulence in different pathogen lineages. Mycobacterium tuberculosis complex taxonomy has rapidly evolved in the last 20 years through intensive clinical isolation, advances in sequencing and in the description of fast-evolving loci (CRISPR and MIRU-VNTR). On-line tools to describe new isolates have been set up based on known diversity either on CRISPRs (also known as spoligotypes) or on MIRU-VNTR profiles. The underlying taxonomies are largely concordant but use different names and offer different depths. The objectives of this study were 1) to explicit the consensus that exists between the alternative taxonomies, and 2) to provide an on-line tool to ease classification of new isolates. Genotyping (24-VNTR, 43-spacers spoligotypes, IS6110-RFLP) was undertaken for 3,454 clinical isolates from the Netherlands (2004-2008). The resulting database was enlarged with African isolates to include most human tuberculosis diversity. Assignations were obtained using TB-Lineage, MIRU-VNTRPlus, SITVITWEB and an algorithm from Borile et al. By identifying the recurrent concordances between the alternative taxonomies, we proposed a consensus including 22 sublineages. Original and consensus assignations of the all isolates from the database were subsequently implemented into an ensemble learning approach based on Machine Learning tool Weka to derive a classification scheme. All assignations were reproduced with very good sensibilities and specificities. When applied to independent datasets, it was able to suggest new sublineages such as pseudo-Beijing. This Lineage Prediction tool, efficient on 15-MIRU, 24-VNTR and spoligotype data is available on the web interface \"TBminer.\" Another section of this website helps summarizing key molecular epidemiological data, easing tuberculosis surveillance. Altogether, we successfully used Machine Learning on a large dataset to set up and make available the first consensual taxonomy for human Mycobacterium tuberculosis complex. Additional developments using SNPs will help stabilizing it.

BACKGROUND: Chloroquine resistance (CR) decreased after the removal of chloroquine from national treatment guidelines in Malawi, Kenia and Tanzania. In this investigation the prevalence of the chloroquine resistance (CQR) conferring mutant pfcrt allele and its associated chromosomal haplotype were determined before and after the change in Gabonese national treatment guidelines from chloroquine (CQ) to artesunate plus amodiaquine (AQ) in 2003.
METHODS: The prevalence of the wild type pfcrt allele was assessed in 144 isolates from the years 2005 - 07 by PCR fragment restriction digest and direct sequencing. For haplotype analysis of the chromosomal regions flanking the pfcrt locus, microsatellite analysis was done on a total of 145 isolates obtained in 1995/96 (43 isolates), 2002 (47 isolates) and 2005 - 07 (55 isolates).
RESULTS: The prevalence of the mutant pfcrt allele decreased from 100% in the years 1995/96 and 2002 to 97% in 2005 - 07. Haplotype analysis showed that in 1995/96 79% of the isolates carried the same microsatellite alleles in a chromosomal fragment spanning 39 kb surrounding the pfcrt locus. In 2002 and 2005 - 07 the prevalence of this haplotype was 62% and 58%, respectively. Pfcrt haplotype analysis showed that all wild type alleles were CVMNK.
CONCLUSIONS: Four years after the withdrawal of CQ from national treatment guidelines the prevalence of the mutant pfcrt allele remains at 97%. The data suggest that the combination of artesunate plus AQ may result in continued selection for the mutant pfcrt haplotype even after discontinuance of CQ usage.

BACKGROUND: Red clover (Trifolium pratense L.) is a major forage legume that has a strong self-incompatibility system and exhibits high genetic diversity within populations. For several crop species, integrated consensus linkage maps that combine information from multiple mapping populations have been developed. For red clover, three genetic linkage maps have been published, but the information in these existing maps has not been integrated.
RESULTS: A consensus linkage map was constructed using six mapping populations originating from eight parental accessions. Three of the six mapping populations were established for this study. The integrated red clover map was composed of 1804 loci, including 1414 microsatellite loci, 181 amplified fragment length polymorphism (AFLP) loci and 204 restriction fragment length polymorphism (RFLP) loci, in seven linkage groups. The average distance between loci and the total length of the consensus map were 0.46 cM and 836.6 cM, respectively. The locus order on the consensus map correlated highly with that of accession-specific maps. Segregation distortion was observed across linkage groups. We investigated genome-wide allele frequency in 1144 red clover individuals using 462 microsatellite loci randomly chosen from the consensus map. The average number of alleles and polymorphism information content (PIC) were 9.17 and 0.69, respectively.
CONCLUSIONS: A consensus genetic linkage map for red clover was constructed for the first time based on six mapping populations. The locus order on the consensus map was highly conserved among linkage maps and was sufficiently reliable for use as a reference for genetic analysis of random red clover germplasms.

BACKGROUND: Molecular marker technologies are undergoing a transition from largely serial assays measuring DNA fragment sizes to hybridization-based technologies with high multiplexing levels. Diversity Arrays Technology (DArT) is a hybridization-based technology that is increasingly being adopted by barley researchers. There is a need to integrate the information generated by DArT with previous data produced with gel-based marker technologies. The goal of this study was to build a high-density consensus linkage map from the combined datasets of ten populations, most of which were simultaneously typed with DArT and Simple Sequence Repeat (SSR), Restriction Enzyme Fragment Polymorphism (RFLP) and/or Sequence Tagged Site (STS) markers.
RESULTS: The consensus map, built using a combination of JoinMap 3.0 software and several purpose-built perl scripts, comprised 2,935 loci (2,085 DArT, 850 other loci) and spanned 1,161 cM. It contained a total of 1,629 \'bins\' (unique loci), with an average inter-bin distance of 0.7 +/- 1.0 cM (median = 0.3 cM). More than 98% of the map could be covered with a single DArT assay. The arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. The locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. The distribution of loci along chromosomes indicated centromeric suppression of recombination in all chromosomes except 5H. DArT markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. On the average, 14 +/- 9 DArT loci were identified within 5 cM on either side of SSR, RFLP or STS loci previously identified as linked to agricultural traits.
CONCLUSIONS: Our barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying DArT markers in molecular breeding schemes. The study also highlights the need for improved software for building consensus maps from high-density segregation data of multiple populations.

To investigate phylogenetic relationships in the genus Cucumis, 9 consensus chloroplast simple sequence repeat (ccSSR) primer pairs (ccSSR3, 9, 11, 13, 14, 17, 20, 21, and 23) were employed for DNA fragment length variation and 5 amplified fragments, ccSSR4, 12, 13, 19, and 20, were sequenced using total DNA from 13 accessions representing 7 African Cucumis species (x = 12), 3 Cucumis melo L. (x = 12) accessions, 2 Cucumis sativus L. (x = 7) accessions, and 1 Cucumis hystrix Chakr. (x = 12) accession. A Citrullus lanatus (Thunb.) Matsum. & Nakai (x = 11) accession was used as an outgroup. While fragment length analysis revealed the existence of 3 major species clusters (i.e., a group of African Cucumis species, a group composed of C. melo accessions, and a group containing C. sativus and C. hystrix species), sequence variation analysis identified 2 major species clusters (i.e., a group of African Cucumis species and a group composed of C. melo, C. sativus, and C. hystrix species). Comparative analysis using nuclear DNA (previous studies) and cpDNA sequence substitution data resulted in the placement of C. melo and C. sativus in different cluster groupings. Thus, both nuclear and cytoplasmic DNA should be employed and compared when a putative progenitor or specimens of an ancestral Cucumis species lineage is investigated. In addition, C. ficifolius (2x) and C. aculeatus (4x) of the African Cucumis species clustered together in this study. This result does not agree with reported isozyme analyses, but does agree with previously characterized chromosome homologies between these 2 species. Although African Cucumis species and C. hystrix do not share a close relationship, genetic affinities between C. sativus and C. hystrix are considerable. Combined evidence from previously published studies and data presented herein lend support to the hypothesis that C. hystrix is either a progenitor species of C. sativus or that they at least share a common ancestral lineage.

OBJECTIVE: To identify 6 major human herpesviruses with consensus primers and to explore its clinical application.
METHODS: Based on the highly-homogeneous regions of DNA polymerase gene in human herpesviruses,Two pairs of primer were synthesized. One pair was designed to amplify herpes simplex virus type 1, type 2, Epstein-Barr virus and cytomegalovirus; and another was used to amplify varicella-zoster virus or human herpesvirus 6. Virus species identification was performed by restriction enzyme digestion with BamH I and BstU I. Thirty-eight CSF specimens of clinically diagnosed viral encephalitis,and 49 blood specimens from 27 confirmed cases and 22 clinically diagnosed ones were tested for herpes virus DNA using the PCR-RFLP assay with these primers.
RESULTS: Thirteen out of 38 CSF specimens (34.2%) were herpes virus positive. All blood specimens from 27 confirmed cases showed positive results, while for 22 clinically diagnosed cases 16 (72.7%) were positive. The types of herpes virus were determined using restriction enzyme digestion with BamH I and BstU I. Two CSF specimens from the patients, who were treated with aciclovir for 2 - 3 days, were still positive for herpes virus DNA by this method. None of the control blood or CSF controls were positive for herpesvirus by PCR.
CONCLUSIONS: The PCR-RFLP method used in this study is a specific, sensitive and practicable one for diagnosis of herpes virus infection.

Eight human viruses of the Herpesviridae family represent a significant public health problem world-wide. Detection and typing of five of the human herpesviruses (HSV-1, HSV-2, VZV, EBV, and CMV) was performed by applying a consensus primer polymerase chain reaction (PCR). The amplified PCR products from the five human herpesviruses were typed based on their restriction enzyme digestion polymorphism with Hinf I and Alu I. Fifteen clinically suspected specimens from herpesvirus-infected patients were also evaluated. A fragment of the DNA polymerase gene from each of the five human herpesviruses was successfully amplified by the set of consensus primers. Their amplicons obtained by PCR from the template DNAs were subjected to restriction endonuclease digestion and human herpesviruses 1-5 could be clearly differentiated and typed. This method can be used to detect and differentiate between the five human herpesviruses in clinical specimens. This study demonstrates the value of testing for five human herpesviruses by consensus PCR and restricted fragment length polymorphism (RFLP). These procedures are simple and straightforward techniques for the investigation of clinical specimens.

BACKGROUND: Hemophilia A, an X-linked recessive disorder, has the prevalence of 1 male per 7000 of the male population in the Northern states of India. The aim of the present study was to analyze the polymorphisms of the factor VIII gene in a sample population comprised of 22 families (112 persons) in order to formulate an algorithm that would be informative and accurate, yet cost-effective for carrier diagnosis.
METHODS: Polymerase chain reaction was used to study the polymorphisms of two intragenic restriction fragment length polymorphic sites (recognized by BclI and HindIII) and an extragenic variable number tandem repeat (VNTR) locus (St14).
RESULTS: Fifty-eight percent of the women tested were heterozygous for the BclI restriction fragment length polymorphism (RFLP) (significantly high compared to earlier reports), signifying the usefulness of this marker in carrier detection. About 64% of the families from the target population could be diagnosed using this marker alone. The other intragenic HindIII RFLP site showed a heterozygosity rate of 43% in women, and was effective in diagnosing 50% of the families studied. The population prevalence for \'+\' alleles of BclI and HindIII were 68% and 33%, respectively. About 88% of the women tested were heterozygous for the St14 locus, and 83% of the families could be diagnosed using this marker alone. The 1390- and 1300-bp alleles were most prevalent, while novel polymorphisms of 1500 and 1345 bp were detected.
CONCLUSIONS: Based on the above data, we suggest screening hemophilic families first for BclI polymorphism, followed by an analysis for HindIII polymorphism in case of homozygosity at the BclI site. When both were noninformative, analysis of St14 locus would be necessary.