Development and evaluation of a quantitative polymerase chain reaction for aquatic Streptococcus agalactiae based on the groEL gene.

AIMS: The aim of this study was to develop a TaqMan quantitative polymerase chain reaction (qPCR), based on the Streptococcus agalactiae groEL gene, to specifically quantify levels of bacteria within samples derived from aquatic sources, particularly aquaculture. Enumeration of bacteria by qPCR was compared with culture-based methods. METHODS AND RESULTS: The qPCR was sensitive to 33 isolates of S. agalactiae, representing 11 clonal complexes from aquatic, bovine and human hosts. The specificity of the assay was 92·5% at a threshold Cq value of 35. No cross-reaction with Streptococcus iniae was noted and of the 22 comparator species screened to test assay specificity, Streptococcus porcinus had a Cq value of 33·7 S, while Streptococcus gallolyticus subsp. macedonicus and Streptococcus ictaluri had one replicate value above the Cq threshold of 35 (34·5 and 34·4 respectively), while only S. agalactiae were detected with a Cq value of 30. The limit of detection of the assay was 1·7 copies per µl at Cq 35. Discrepancies between molecular and culture-based methods of enumeration were noted. CONCLUSIONS: The qPCR was able to detect a diverse range of S. agalactiae isolates from different clonal complexes (CCs) and could distinguish between S. agalactiae and closely related species, notably S. iniae. The results suggest that a Cq 30 would be a very meaningful cut-off, allowing the detection of infected fish while ruling out all false positives. SIGNIFICANCE AND IMPACT OF THE STUDY: This rapid and sensitive qPCR assay is useful to quantify DNA copy number in the laboratory and could prove useful for detecting low levels of S. agalactiae in aquaculture systems, including Oreochromis niloticus culture.

Evaluation of PCR primers targeting the groEL gene for the specific detection of Streptococcus agalactiae in the context of aquaculture.

AIMS: The aim of this study was to design a set of primers for specific detection and identification of Streptococcus agalactiae in polymerase chain reaction (PCR) that can detect a diverse range of S. agalactiae isolates from different hosts and that it is capable of discriminating between S. agalactiae and other species that are closely related or potentially present in aquaculture environments, notably Streptococcus iniae. METHODS AND RESULTS: Primers, based on the groEL2 gene of S. agalactiae, were shown to be epidemiologically sensitive to 97 isolates of S. agalactiae, representing 11 clonal complexes derived from piscine, terrestrial and aquatic mammalian host species. The primers were tested with 10 S. iniae isolates and 22 other comparator species with no cross-reaction observed after optimization of reaction conditions. They have a high analytical sensitivity, detecting as few as 10 copies of S. agalactiae genomic DNA per reaction and are capable of detecting the target in DNA extracted from the brains of infected fish. CONCLUSIONS: The primers proved suitable for the sensitive and specific detection of S. agalactiae from dairy-, human- and fish-related origins by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the importance of S. agalactiae as a pathogen, many PCR primers have been published for this bacterium, designed largely for its detection in dairy and human samples, but many cross-reacting with S. iniae. The ability to differentiate between S. agalactiae and S. iniae in aquaculture derived samples is important as both infect fish, causing similar disease symptoms and are phenotypically similar, yet control strategies and zoonotic risk are species specific.

Radiation litigation and the nuclear industry--the experience in the United Kingdom.

In the United Kingdom, the Nuclear Installations Act 1965 places a "strict" statutory duty on the operators of nuclear facilities to ensure that any exposure to radiation resulting from operations does not cause injury or damage. A claimant does not have to prove fault to receive compensation under the Act, only causation. The 1965 Act has been fundamental in shaping litigation involving the nuclear industry in the UK. Civil law cases brought under the Act will be heard before a single judge (with no jury or technical assessor) who must present his or her decision in a reasoned judgment. This process leads to a considerable volume of expert evidence being presented to the court and extensive cross-examination of witnesses. The expense and uncertain outcome of cases involving claims by nuclear workers that occupational exposure to radiation had caused the development of cancer has led to employers and trade unions setting up the voluntary Compensation Scheme for Radiation-linked Diseases as an alternative to litigation. This Scheme has worked well and is held up as a model of alternative dispute resolution. However, a few cases concerning personal injury or damage to property have come before the courts when the defendant nuclear operator considered that the claims were technically unjustified and where settlement was not a policy option. As anticipated, these cases were lengthy, complex, and expensive. The radiation doses assessed to have been received by the individuals who were the subject of claims, whether workers or members of the public, have been crucial to the outcome. The technical expertise of health physicists and allied specialists has been vital in establishing defensible estimates of dose, and this contribution can be expected to remain of high importance in radiation litigation in the UK.

A review of probability of causation and its use in a compensation scheme for nuclear industry workers in the United Kingdom.

The assumption that any additional exposure to ionizing radiation leads to an increase in the risk of stochastic health effects implies that some cases of these effects will be caused by exposure incurred occupationally. The main health effect expected to arise in the exposed individuals is cancer. Such radiation-induced cancers cannot be distinguished from the far larger number of background cancers, and, therefore, causation must be assessed statistically. The probability of causation methodology has been developed to ascertain the likelihood that a particular cancer may be attributed to a particular prior exposure to radiation. Given the pertinent details of an individual case, a probability of causation value is calculated from the appropriate relative risk obtained from radiation risk models derived from the epidemiological study of exposed populations, although there are many uncertainties inherent in a particular probability of causation calculation. In the United Kingdom, the Compensation Scheme for Radiation-linked Diseases has been created to determine whether those individuals occupationally exposed to radiation in the nuclear industry who have subsequently developed a malignant disease should be compensated. The Scheme is a voluntary arrangement based upon the probability of causation methodology, which incorporates various procedures agreed by employer and employee representatives and their advisers. In a pragmatic approach to compensation, the uncertainties of a specific probability of causation calculation are accommodated through generosity factors which favor the claimant and encourage the use of the Scheme. The Scheme, which was introduced in 1982 and modified in 1987 and 1991 in the light of operational experience and revised risk estimates, has provided a successful alternative to litigation from the point of view of both employer and employee.

Substituent effects on the reactivity of the silicon-carbon double bond.

Laser flash photolysis of various organosilicon compounds such as aryl-, vinyl-, and alkynyldisilanes, silacyclobutanes and silacyclobutenes, and alpha-silylketenes and -diazomethanes leads to the formation of reactive silenes which can be detected directly in solution, allowing detailed studies of the kinetics and mechanisms of their reactions with nucleophiles. Over 30 transient silenes have now been studied by these methods, providing the opportunity to systematically assess the effects of substituents at silicon and carbon on the reactivity of the Si=C bond.

Conrotatory photochemical ring opening of alkylcyclobutenes in solution. A test of the hot ground-state mechanism.

Quantum yields for photochemical ring opening of six alkylcyclobutenes have been measured in hexane solution using 228-nm excitation, which selectively populates the lowest pi,R(3s) excited singlet states of these molecules and has been shown previously to lead to ring opening with clean conrotatory stereochemistry. The compounds studied in this work-1,2-dimethylcyclobutene (1), cis- and trans-1,2,3,4-tetramethylcyclobutene (cis- and trans-5), hexamethylcyclobutene (8), and cis- and trans-tricyclo[6.4.0.0(2,7)]dodec-1(2)-ene (cis- and trans-9)-were selected so as to span a broad range in molecular weight and as broad a range as possible in Arrhenius parameters for thermal (ground-state) ring opening. RRKM calculations have been carried out to provide estimates of the rate constants for ground-state ring opening of each of the compounds over a range of thermal energies from 20 000 to 49 000 cm(-1). These have been used to estimate upper limits for the quantum yields of ring opening via a hot ground-state mechanism, assuming a value of k(deact) = 10(11) s(-1) for the rate constant for collisional deactivation by the solvent, that internal conversion to the ground state from the lowest Rydberg state occurs with close to unit efficiency, and that ergodic behavior is followed. The calculated quantum yields are significantly lower than the experimental values in all cases but one (1). This suggests that the Rydberg-derived ring opening of alkylcyclobutenes is a true excited-state process and rules out the hot ground-state mechanism for the reaction.