Communal larval rearing of European lobster (Homarus gammarus): family identification by microsatellite DNA profiling an offspring fitness.

Stock enhancement experiments of European lobster (Homarus gammarus) have been carried out around the Kvitsoy Islands in south-western Norway since 1990. In addition to releases of coded wire tagged lobster juveniles (cultured) and subsequent monitoring of commercial fishery, a lobster hatchery was established in 1997. Several experiments were made on the communal-rearing approach where the performance of mixed larval groups (families) was evaluated under identical conditions. Berried females of wild and cultured origin and their respective fertilised eggs were screened by using microsatellite DNA profiling involving a multiplex set of six lobster specific primers, thereby allowing determination of both parental genotypes. Each female were kept separately during hatching, and the offspring were later mixed and raised in a communal rearing system. The early-larval survival was estimated at stage IV (bottom stage), and the survivors were identified to family and group by microsatellite profiling. Five different communal experiments were conducted, representing offspring from 65 berried females. Of the surviving larvae, 6.3% could not be assigned to family due to degraded DNA and no PCR amplification. Significant differences in early survival between offspring of wild and cultured origin were found in the experiments. No differences between the groups were found in stage IV larval size. Based on the pooled data on survival (as a measure of early larvae fitness) offspring of cultured females displayed a relative fitness of 60% in comparison to offspring from wild females. Large variation in survival was also observed among families within the wild and cultured groups, suggesting a genetic component for these traits and a potential for selective breeding.

Improving molecular detection of Candida DNA in whole blood:comparison of seven fungal DNA extraction protocols using real-time PCR

The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.

Detection of avian influenza virus by fluorescent DNA barcode-based immunoassay with sensitivity comparable to PCR

In this paper, a coupling of fluorophore-DNA barcode and bead-based immunoassay for detecting avian influenza virus (AIV) with PCR-like sensitivity is reported. The assay is based on the use of sandwich immunoassay and fluorophore-tagged oligonucleotides as representative barcodes. The detection involves the sandwiching of the target AIV between magnetic immunoprobes and barcode-carrying immunoprobes. Because each barcode-carrying immunoprobe is functionalized with a multitude of fluorophore-DNA barcode strands, many DNA barcodes are released for each positive binding event resulting in amplification of the signal. Using an inactivated H16N3 AIV as a model, a linear response over five orders of magnitude was obtained, and the sensitivity of the detection was comparable to conventional RT-PCR. Moreover, the entire detection required less than 2 hr. The results indicate that the method has great potential as an alternative for surveillance of epidemic outbreaks caused by AIV, other viruses and microorganisms.

Detection of Avian Influenza Virus with PCR-Like Sensitivity by Fluorescent DNA Barcode-Based Immunoassay

In this paper, we report a coupling of fluorophore-DNA barcode and bead-based
immunoassay for the detection of Avian Influenza Virus (AIV), a potential pandemic threat for human health and enormous economic losses. The detection strategy is based on the use of sandwich immunoassay and fluorophore-tagged oligonucleotides as representatively fluorescent barcodes. Despite its simplicity the assay has sensitivity comparable to RT-PCR amplification, and possesses a great potential as a rapid and sensitive on-chip detection format.

Diagnostic accuracy of loop-mediated isothermal amplification as a near-patient test for meningococcal disease in children: An observational cohort study

Background: Diagnosis of meningococcal disease relies on recognition of clinical signs and symptoms that are notoriously non-specific, variable, and often absent in the early stages of the disease. Loop-mediated isothermal amplification (LAMP) has previously been shown to be fast and effective for the molecular detection of meningococcal DNA in clinical specimens. We aimed to assess the diagnostic accuracy of meningococcal LAMP as a near-patient test in the emergency department.

Methods: For this observational cohort study of diagnostic accuracy, children aged 0-13 years presenting to the emergency department of the Royal Belfast Hospital for Sick Children (Belfast, UK) with suspected meningococcal disease were eligible for inclusion. Patients underwent a standard meningococcal pack of investigations testing for meningococcal disease. Respiratory (nasopharyngeal swab) and blood specimens were collected from patients and tested with near-patient meningococcal LAMP and the results were compared with those obtained by reference laboratory tests (culture and PCR of blood and cerebrospinal fluid).

Findings: Between Nov 1, 2009, and Jan 31, 2012, 161 eligible children presenting at the hospital underwent the meningococcal pack of investigations and were tested for meningococcal disease, of whom 148 consented and were enrolled in the study. Combined testing of respiratory and blood specimens with use of LAMP was accurate (sensitivity 89% [95% CI 72-96], specificity 100% [97-100], positive predictive value 100% [85-100]; negative predictive value 98% [93-99]) and diagnostically useful (positive likelihood ratio 213 [95% CI 13-infinity] and negative likelihood ratio 0·11 [0·04-0·32]). The median time required for near-patient testing from sample to result was 1 h 26 min (IQR 1 h 20 min-1 h 32 min).

Interpretation: Meningococcal LAMP is straightforward enough for use in any hospital with basic laboratory facilities, and near-patient testing with this method is both feasible and effective. By contrast with existing UK National Institute of Health and Care Excellence guidelines, we showed that molecular testing of non-invasive respiratory specimens from children is diagnostically accurate and clinically useful.

PCR amplification of short tandem repeat sequences allows serial studies of chimaerism/engraftment following BMT in rodents

Animal models of bone marrow transplantation (BMT) allow evaluation of new experimental treatment strategies. One potential strategy involves the treatment of donor marrow with ultra-violet B light to allow transplantation across histocompatibility boundaries without an increase in graft rejection or graft-versus-host disease. A major requirement for a new experimental protocol, particularly if it involves manipulation of the donor marrow, is that the manipulated marrow gives rise to long-term multilineage engraftment. DNA based methodologies are now routinely used by many centres to evaluate engraftment and degree of chimaerism post-BMT in humans. We report the adaptation of this methodology to the serial study of engraftment in rodents. Conditions have been defined which allow analysis of serial tail vein samples using PCR of short tandem repeat sequences (STR-PCR). These markers have been used to evaluate the contribution of ultraviolet B treated marrow to engraftment following BMT in rodents without compromising the health of the animals under study. Chimaerism data from sequential tail vein samples and bone marrow from selected sacrificed animals showed excellent correlation, thus confirming the validity of this approach in analysing haemopoietic tissue. Thus the use of this assay may facilitate experimental studies in animal BMT.

Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: A duplex approach with DNA binding dye chemistry

Schistosomiasis is a chronically debilitating helminth infection with a significant socio-economic and public health impact. Accurate diagnostics play a pivotal role in achieving current schistosomiasis control and elimination goals. However, many of the current diagnostic procedures, which rely on detection of schistosome eggs, have major limitations including lack of accuracy and the inability to detect pre-patent infections. DNA-based detection methods provide a viable alternative to the current tests commonly used for schistosomiasis diagnosis. Here we describe the optimisation of a novel droplet digital PCR (ddPCR) duplex assay for the diagnosis of Schistosoma japonicum infection which provides improved detection sensitivity and specificity. The assay involves the amplification of two specific and abundant target gene sequences in S. japonicum; a retrotransposon (SjR2) and a portion of a mitochondrial gene (nad1). The assay detected target sequences in different sources of schistosome DNA isolated from adult worms, schistosomules and eggs, and exhibits a high level of specificity, thereby representing an ideal tool for the detection of low levels of parasite DNA in different clinical samples including parasite cell free DNA in the host circulation and other bodily fluids. Moreover, being quantitative, the assay can be used to determine parasite infection intensity and, could provide an important tool for the detection of low intensity infections in low prevalence schistosomiasis-endemic areas.