An efficient and novel technology for the extraction of parasite genomic DNA from whole blood or culture

The aim of this study was to assess pathogen DNA extraction with a new spin column-based method (DNA-XT). DNA from either whole-blood samples spiked with Plasmodium falciparum or Leishmania donovani amastigote culture was extracted with DNA-XT and compared with that produced by a commercial extraction kit (DNeasy®). Eluates from large and small sample volumes were assessed by PCR and spectroscopy. Using a small volume (5 μl) of blood, the DNA-XT and DNeasy methods produced eluates with similar DNA concentrations (0.63 vs 1.06 ng/μl, respectively). The DNA-XT method produced DNA with lower PCR inhibition than DNeasy. The new technique was also twice as fast and required fewer plastics and manipulations but had reduced total recovered DNA compared with DNeasy

Identification of Serotype in Culture Negative Pneumococcal Meningitis Using Sequential Multiplex PCR: Implication for Surveillance and Vaccine Design

BACKGROUND: PCR-based serotyping of Streptococcus pneumoniae has been proposed as a simpler approach than conventional methods, but has not been applied to strains in Asia where serotypes are diverse and different from other part of the world. Furthermore, PCR has not been used to determine serotype distribution in culture-negative meningitis cases. METHODOLOGY: Thirty six serotype-specific primers, 7 newly designed and 29 previously published, were arranged in 7 multiplex PCR sets, each in new hierarchies designed for overall serotype distribution in Bangladesh, and specifically for meningitis and non-meningitis isolates. Culture-negative CSF specimens were then tested directly for serotype-specific sequences using the meningitis-specific set of primers. PCR-based serotyping of 367 strains of 56 known serotypes showed 100% concordance with quellung reaction test. The first 7 multiplex reactions revealed the serotype of 40% of all, and 31% and 48% non-meningitis and meningitis isolates, respectively. By redesigning the multiplex scheme specifically for non-meningitis or meningitis, the quellung reaction of 43% and 48% of respective isolates could be identified. Direct examination of 127 culture-negative CSF specimens, using the meningitis-specific set of primers, yielded serotype for 51 additional cases. CONCLUSIONS: This PCR approach, could improve ascertainment of pneumococcal serotype distributions, especially for meningitis in settings with high prior use of antibiotics

Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods

To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit MGrade), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay

Molecular Typing of Treponema pallidum: A Systematic Review and Meta-Analysis

Syphilis has been resurgent in many parts of the world in past decades. Understanding the epidemiology of syphilis is important for estimating disease burdens, monitoring epidemic trends, and evaluating intervention activities. Treponema pallidum (T. pallidum), the pathogen of syphilis, cannot be grown in vitro. Because T. pallidum cannot be cultured, molecular typing of T. pallidum is particularly useful and allows for investigation of infection diversity and epidemiology. We conducted a statistical analysis of available published data to investigate the current research progress of molecular typing of syphilis. Our analysis showed that primary lesion was a better specimen for obtaining T. pallidum DNA than blood. Blood specimens collected from scraping the ear lobes had high yield of T. pallidum DNA and high full typing efficiency. Ear lobe blood is a promising specimen for future T. pallidum molecular typing, but further research should verify this finding using a larger sample size. Within all studies, subtype 14d was most prevalent, and subtype distribution varied across geographic areas. Subtype data associated with macrolide resistance and neurosyphilis were limited. More research on molecular typing of T. pallidum can be useful for investigating syphilis epidemiology and designing syphilis control strategies

Universal Primers Used for Species Identification of Foodstuff of Animal Origin: Effects of Oligonucleotide Tails on PCR Amplification and Sequencing Performance

M13 universal non-homologous oligonucleotide tails incorporated into universal primers have been shown to improve amplification and sequencing performance. However, a few protocols use these tails in the field of food inspection. In this study, two types of M13 tails (by Steffens and Messing) were selected to assess their benefits using universal cytochrome oxidase subunit I (COI) and 16S ribosomal RNA gene (16SrRNA) primers in standard procedures. The primer characteristics were tested in silico. Then, using 20 DNA samples of edible species (birds, fishes, and mammals), their performance during PCR amplification (band recovery and intensity) and sequencing (sequence recovery, length, and Phred score) was assessed and compared. While 16SrRNA tailed and non-tailed primers performed similarly, differences were found for COI primers. Messing’s tails negatively affected the reaction outputs, while Steffens’ tails significantly improved the band intensity and the length of the final contigs based on the individual bidirectional read sequence. This different performance could be related to a destabilization effect of certain tails on primers with unfavorable mismatches on the annealing region. Even though our results cannot be generalized because the tail performances are strictly dependent on laboratory conditions, they show that appropriate tails can improve the overall throughput of the analysis, supporting food traceabilit

Circulating miRNA signatures of early pregnancy in cattle

BACKGROUND: Low fertility remains a leading cause of poor productivity in dairy cattle. In this context, there is significant interest in developing novel tools for accurate early diagnosis of pregnancy. MicroRNAs (miRNAs) are short RNA molecules which are critically involved in regulating gene expression during both health and disease. MiRNAs have been shown to regulate ovarian function, uterine receptivity, embryonic development and placental function. Circulating miRNAs can provide useful biomarkers of tissue function and disease; importantly, differential miRNA profiles have been linked to pregnancy and preeclampsia in humans. This study sought to establish the potential of circulating miRNAs as biomarkers of early pregnancy in cattle. RESULTS: We applied Illumina small-RNA sequencing to profile miRNAs in plasma samples collected from eight non-pregnant heifers on Days 0, 8 and 16 of the oestrous cycle and 11 heifers on Days 16 and 24 of pregnancy. We sequenced a total of 46 samples and generated 9.2 million miRNA reads per sample. There were no differences in miRNA read abundance between any of the pregnant and non-pregnant time-points (FDR > 0.1). As a complementary approach, we analysed sample pools (3–4 samples/pool) corresponding to Days 0, 8 and 16 of the oestrous cycle and Day 24 of pregnancy (n = 3 pools/group) using Qiagen PCR arrays. A total of 16 miRNAs were differentially expressed (FDR < 0.1) in plasma between pregnant and non-pregnant animals. RT-qPCR validation using the same plasma samples confirmed that miR-26a was differentially upregulated on Day 16 pregnant relative to non-pregnant heifers (1.7-fold; P = 0.043), whereas miR-1249 tended to be upregulated in Day 16 pregnant heifers (1.6-fold; P = 0.081). Further validation in an independent group of heifers confirmed an increase in plasma miR-26a levels during early pregnancy, which was significant only on Day 24 (2.0-fold; P = 0.027). CONCLUSIONS: Through genome-wide analyses we have successfully profiled plasma miRNA populations associated with early pregnancy in cattle. We have identified miR-26a as a potential circulating biomarker of early pregnancy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2529-1) contains supplementary material, which is available to authorized users