Measuring Digital PCR Quality: Performance Parameters and Their Optimization.

Digital PCR is rapidly being adopted in the field of DNA-based food analysis. The direct, absolute quantification it offers makes it an attractive technology for routine analysis of food and feed samples for their composition, possible GMO content, and compliance with labelling requirements. However, assessing the performance of dPCR assays is not yet well established. This article introduces three straightforward parameters based on statistical principles that allow users to evaluate if their assays are robust. In addition, we present post-run evaluation criteria to check if quantification was accurate. Finally, we evaluate the usefulness of Poisson confidence intervals and present an alternative strategy to better capture the variability in the analytical chain.

Use of pJANUS-02-001 as a calibrator plasmid for Roundup Ready soybean event GTS-40-3-2 detection: an interlaboratory trial assessment.

Owing to the labelling requirements of food and feed products containing materials derived from genetically modified organisms, quantitative detection methods have to be developed for this purpose, including the necessary certified reference materials and calibrator standards. To date, for most genetically modified organisms authorized in the European Union, certified reference materials derived from seed powders are being developed. Here, an assessment has been made on the feasibility of using plasmid DNA as an alternative calibrator for the quantitative detection of genetically modified organisms. For this, a dual-target plasmid, designated as pJANUS-02-001, comprising part of a junction region of genetically modified soybean event GTS-40-3-2 and the endogenous soybean-specific lectin gene was constructed. The dynamic range, efficiency and limit of detection for the soybean event GTS-40-3-2 real-time quantitative polymerase chain reaction (Q-PCR) system described by Terry et al. (J AOAC Int 85(4):938-944, 2002) were shown to be similar for in house produced homozygous genomic DNA from leaf tissue of soybean event GTS-40-3-2 and for plasmid pJANUS-02-001 DNA backgrounds. The performance of this real-time Q-PCR system using both types of DNA templates as calibrator standards in quantitative DNA analysis was further assessed in an interlaboratory trial. Statistical analysis and fuzzy-logic-based interpretation were performed on critical method parameters (as defined by the European Network of GMO Laboratories and the Community Reference Laboratory for GM Food and Feed guidelines) and demonstrated that the plasmid pJANUS-02-001 DNA represents a valuable alternative to genomic DNA as a calibrator for the quantification of soybean event GTS-40-3-2 in food and feed products.

Measles outbreak in a fully immunized secondary-school population.

An outbreak of measles occurred among adolescents in Corpus Christi, Texas, in the spring of 1985, even though vaccination requirements for school attendance had been thoroughly enforced. Serum samples from 1806 students at two secondary schools were obtained eight days after the onset of the first case. Only 4.1 percent of these students (74 of 1806) lacked detectable antibody to measles according to enzyme-linked immunosorbent assay, and more than 99 percent had records of vaccination with live measles vaccine. Stratified analysis showed that the number of doses of vaccine received was the most important predictor of antibody response. Ninety-five percent confidence intervals of seronegative rates were 0 to 3.3 percent for students who had received two prior doses of vaccine, as compared with 3.6 to 6.8 percent for students who had received only a single dose. After the survey, none of the 1732 seropositive students contracted measles. Fourteen of 74 seronegative students, all of whom had been vaccinated, contracted measles. In addition, three seronegative students seroconverted without experiencing any symptoms. We conclude that outbreaks of measles can occur in secondary schools, even when more than 99 percent of the students have been vaccinated and more than 95 percent are immune.

Specific immunoglobulin M enzyme-linked immunosorbent assay for confirming the diagnosis of measles.

An enzyme-linked immunosorbent assay (ELISA) was evaluated for the detection of measles virus-specific immunoglobulin M (IgM) (MIgM). The ELISA was standardized by deriving a seronegative range of values from sera which should not contain MIgM (24 cord sera, 59 sera from immune health care workers, and 47 sera from infants before the administration of measles vaccine). These values were separable from those obtained from individuals convalescing from measles. Twenty sera containing rheumatoid factor were MIgM seronegative. Of 30 acute-phase sera from suspected measles cases, 26 contained MIgM; those that were seronegative were obtained on day 0, 0, 2, or 9. All 25 convalescent-phase samples contained MIgM. Of the 25 paired samples, 22 were IgG positive at the first sampling; 3 of the 22 did not show a rise in IgG titer. The MIgM ELISA can be used for confirming suspected measles cases, often requiring only a single serum specimen.

Varicella in day care centers.

Spread of varicella in day care is controlled by excluding children at the first signs of illness. Exclusion is generally ineffective. Minimally ill children might be permitted to attend day care. This approach may lead to exposure of those in high-risk groups, i.e., adults or immunocompromised children. Morbidity is greater in adults, but susceptibility among day care workers is probably low. Immunocompromised children can be vaccinated or given varicella-zoster immune globulin (VZIG) after exposure. Questions about the risks of varicella-zoster vaccination (V-Z) concern the production of latent infection and subsequent zoster and the effect on the epidemiology of infection, i.e., its possible delay until adulthood. Zoster has been found not to be more frequent in immunized than in nonimmunized leukemic children, and normal vaccinees retain good antibody titers five years after vaccination and have titers similar to individuals who have had varicella 10 years after vaccination. Vaccine efficacy is excellent, but its desirability will be determined after resolution of questions concerning the long-term impact of its use.

Impact of revaccinating children who initially received measles vaccine before 10 months of age.

Two hundred fifty-four infants who had received measles vaccine at less than 10 months of age were revaccinated at greater than or equal to 15 months of age, and their immune responses were compared with 129 control infants who received their first doses of measles vaccine at greater than or equal to 15 months of age. Sera were collected at the time of revaccination (study infants) or primary vaccination (control infants), 3 weeks, and 8 months later and tested for antibody by hemagglutination inhibition (HI), enzyme-linked immunosorbent assay (ELISA), and cytopathic effect neutralization (CPEN). Of the 121 study infants who were initially HI negative, 116 (95.9%) made HI antibody 3 weeks postrevaccination compared with 126 (99.2%) of 127 control infants (P = 0.19). Of the 63 study infants with no initial detectable antibody by any of the three tests, 14 (22.2%) had a measles-specific IgM response 3 weeks postrevaccination compared with 37 of 50 (74.0%) randomly chosen control infants. By 8 months after revaccination, the 121 initially HI-negative study infants were significantly less likely to have detectable HI antibodies than control infants (52.1% v 97.6%) (P less than .001). However, 96.7% of these 121 study infants had detectable neutralizing antibody 8 months postrevaccination, an antibody thought to correlate best with protection. This study confirms the altered immune response to revaccination in infants first vaccinated prior to 10 months of age; however, the data suggest that most of these infants were successfully primed and are probably protected after revaccination.

Effect of early immunization on antibody response to reimmunization with measles vaccine as demonstrated by enzyme-linked immunosorbent assay (ELISA).

A measles epidemic in San Antonio, Texas provided a population of children who were immunized at less than or equal to 10 months of age and reimmunized at greater than or equal to 15 months of age. Of these children, 302 were evaluated for measles antibody by the sensitive enzyme-linked immunosorbent assay (ELISA), and their responses were compared with those of 300 children who had been immunized at the customary time (greater than or equal to 15 months) with a single immunization. There were only five seronegative findings in each group. The children immunized at the customary time did have significantly higher (P less than .001) antibody titers than the children immunized at less than or equal to 10 months and reimmunized at greater than or equal to 15 months. These results indicate that early immunization followed by reimmunization may be indicated when young infants are at significant risk of measles exposure. This approach should not create an increased number of serologically nonresponsive children when reimmunized at greater than or equal to 15 months.