Genotyping of the reduced folate carrier‐1 c.80G>A polymorphism by pyrosequencing technology: Importance of PCR and pre‐PCR optimization

When developing a genotyping assay by Pyrosequencing^? technology for the RFC1 (SLC19A1) c.80G>A polymorphism (rs1051266), unequal peak heights in the pyrograms were observed, probably due to unequal amplification of the mutated and wild‐type alleles. This rarely occurring problem could potentially render assignment of heterozygous genotypes uncertain. When the PCR conditions were studied, it was found that substitution of the dGTP nucleotide in the master mix by dGTP and dITP in proportion 1:1 largely overcame this problem. Heat denaturation of the DNA at 95°C before PCR also counteracted the problem. A combination of these two modifications of the standard pyrosequencing PCR protocol gave the best results. We conclude that, with these modifications, the RFC1 c.80G>A SNP can be reliably assayed by pyrosequencing.     

ALIS‐FLP: Amplified ligation selected fragment‐length polymorphism method for microbial genotyping

A DNA fingerprinting method known as ALIS‐FLP (amplified ligation selected fragment‐length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs in that only one specific restriction enzyme (TspRI) is used. The cohesive ends of the DNA fragments are ligated with two types of oligonucleotide. A long oligonucleotide containing the primer site and the specific 9 nt 3 prime end, which is complementary to specific 9 nt, cohesive 3 prime end of the TspRI genomic DNA fragment, and a short, degenerated, oligonucleotide covering the remaining TspRI cohesive ends. Other cohesive ends are covered by a short degenerated oligonucleotide lacking the primer site. The ligation mixture is used as a template for amplification using a single primer corresponding to the 5 prime end of the long, specific oligonucleotide. The selection of TspRI digested genomic DNA fragments for amplification is achieved by sequence selective ligation of the specific long oligonucleotide carrying the primer site to both ends of the specific target fragment. This technique allows for differentiation of the organisms without previous knowledge of their DNA sequence. The usefulness of the method is confirmed by genotyping of 70 previously characterized clinical E. coli isolates. The grouping obtained was identical to the results of REA‐PFGE. Versatility of the method is highlighted, i.e. its combining the advantages of the AFLP technique with a simple, rapid and cheap polymerase chain reaction product detection method.     

Histamine and Leukocytes in Blood During Muscular Work in Man

It has been reported that flow cytometry can be used as a reference procedure to determine sperm concentrations in quality control schemes in andrology laboratories, but there are no convincing quality control data. To understand comprehensively whether flow cytometry can be used to determine sperm concentration, sperm concentrations of 85 human semen samples were detected using three different methods, namely flow cytometry, computer‐assisted semen analysis (CASA) and manual counting with a cell‐VU chamber. The bead concentrations of both low [(18±2.5)×10⁶/mL] and high [(35±5)×10⁶/mL] pre‐calibrated standard latex bead solutions were also determined with flow cytometry. The results showed that bead concentrations of both low and high pre‐calibrated standard latex bead solutions counted five times with flow cytometry were (21.37±0.85)×10⁶/mL and (45.95±1.76)×10⁶/mL, respectively. Coefficient variances (CVs) and relative errors (REs) were 4%, 15.51% and 3.84%, 31.3% for low and high latex bead solutions, respectively. The overall correlation between values measured with flow cytometry and values measured with the cell‐VU chamber and the CASA system was significant. However, flow cytometry overestimated the sperm concentration by 109% compared to the results with the cell‐VU chamber. Moreover, for the azoospermic samples analysed, the sperm concentration was estimated at 0.12 (range from 0.04 to 0.24)×10⁶/mL. In conclusion, the data demonstrated that flow cytometry can result in an overestimation of both bead counting and sperm concentration, suggesting that flow cytometry is an inappropriate method for sperm counting, especially in the case of azoospermia.     

Comparison of allergen-specific IgE levels between Immulite 2000 and ImmunoCAP systems against six inhalant allergens and ten food allergens

Abstract

In vitro allergen-specific immunoglobulin E (sIgE) measurement has been used as an important diagnostic tool for allergic diseases. Currently, quantitative sIgE levels are detected mainly by using ImmunoCAP and Immulite 2000 assay system. These two systems have the same calibration scale at 0–100 kUA/L, but they differ in used allergens, detection methods and automation systems. We compared 1600 paired sIgE results for 204 allergic patients, including 100 paired sIgE assay results for each of 16 allergens (Alternaria alternata, birch-alder mix, cat dander, D. farinae, D. pteronyssinus, dog dander, buckwheat, crab, egg white, mackerel, milk, peach, peanut, shrimp, soybean and wheat flour). Inter-method comparison was performed for qualitative data with a cutoff of 0.35 kUA/L and a detection limit of 0.1 kUA/L, semi-quantitative class results and quantitative data. In qualitative comparisons, the overall concordance rate ranged from 81.0% to 99.0% (k: 0.599–0.949) with the cutoff value of 0.35 kUA/L. It also ranged from 80.0% to 99.0% (k: 0.521–0.951) with the detection limit of 0.1 kUA/L. The class results from these two assays showed good agreements for all allergens. For quantitative sIgE results, these two assays showed moderate positive correlations for Dog dander (r?=?0.683) and Mackerel (r?=?0.573) but high to very high correlations for the other 14 allergens (r?=?0.734–0.972). Immulite 2000 and ImmunoCAP assays demonstrated good concordance and correlation for 16 common allergens, but international standards against each specific allergen for calibration and harmonization of sIgE tests are still needed.