Comparison of ligase detection reaction and real-time PCR for detection of low abundant YMDD mutants in patients with chronic hepatitis B.

AIM: To compare the ligase detection reaction (LDR) and real-time PCR for detection of low abundant YMDD mutants in patients with chronic hepatitis B infection. METHODS: Mixtures of plasmids and serum samples from 52 chronic hepatitis B patients with low abundant lamivudine-resistant mutations were tested with LDR and real-time PCR. Time required and reagent cost for both assays were evaluated. RESULTS: Real-time PCR detected 100, 50, 10, 1 and 0.1% of YIDD plasmid, whereas LDR detected 100, 50, 10, 1, 0.1, and 0.01% of YIDD plasmid, in mixtures with YMDD plasmid of 10(6) copies/mL. Among the 52 clinical serum samples, completely concordant results were obtained for all samples by both assays, and 39 YIDD, 9 YVDD, and 4 YIDD/YVDD were detected. Cost and time required for LDR and real-time PCR are 60/80 CNY (8/10.7 US dollars) and 4.5/2.5 h, respectively. CONCLUSION: LDR and real-time PCR are both sensitive and inexpensive methods for monitoring low abundant YMDD mutants during lamivudine therapy in patients with chronic hepatitis B. LDR is more sensitive and less expensive, while real-time PCR is more rapid.

Comparison of amplicon-sequencing, pyrosequencing and real-time PCR for detection of YMDD mutants in patients with chronic hepatitis B.

AIM: To compare the sequencing of PCR products, pyrosequencing, and real-time PCR for detection of Tyrosine-methionine-aspartate-aspartate (YMDD) mutants in patients with chronic hepatitis B. METHODS: Mixtures of plasmids and serum samples from 69 chronic hepatitis B patients treated with lamivudine were tested for YMDD mutations by sequencing of PCR products, pyrosequencing, and real-time PCR, respectively. Time required and reagent costs of the three assays were evaluated. RESULTS: Real-time PCR detected 100%, 50%, 10%, 1% and 0.1% of YVDD plasmid in mixtures with 10(6) copies/mL of YMDD plasmid, whereas sequencing and pyrosequencing only detected 100% and 50% of YVDD plasmid in aliquots of the corresponding mixtures. Completely concordant results were obtained from 60 (87%) out of the 69 clinical serum samples by the three assays. Mutants were detected by real-time PCR in less than 20% of the total virus population, but no mutant was detected by sequencing and pyrosequencing. In addition, real-time PCR required less time and was more cost-effective than the other two assays. However, throughput of pyrosequencing was the highest. CONCLUSION: Among the three assays compared, real-time PCR is the most sensitive, cost-effective, and time saving for monitoring YMDD mutants in patients with chronic hepatitis B on lamivudine therapy.

[Analysis of the probiotic Bifidobacterium and Lactobacillus community in child intestinal flora].

To investigate the distribution of child intestinal flora and the composition of its key probiotics community, study on intestinal flora of 21 Chinese children (age 2 - 5) was conducted, which included bacteria isolation and counting, 16S rDNA sequencing and homology analysis. For identification of the key probiotics such as Bifidobacterium and Lactobacillus in children feces at the species level, the specific primers Im26/Im3 and L159/L677 for PCR amplification of partial 16S rDNA were used. The results show that the composition of child intestinal flora is was relatively stable and almost same to the intestinal flora of the youth (age 20 - 25). Culture-based approaches show that the key probiotic community in feces at the species level was highly different in composition and numbers from individual to individual. B. longum and B. pseudocatenulatum, which are detected at levels of 10(7) CFU/g (wet) in samples and the detection rates are 90.48% and 85.71% respectively, are believed to be major bifidobacterial species in child intestinal microbiota. In addition, B. adolescentis, B. bifidum, B. infantis and B. thermacidophium have also been found. L. mucosae, L. fermentum, L. salivarius, L. ruminis, L. gasseri and L. plantarum are isolated from the stools. L. mucosae (3.68 log10 CFU/g (wet), detection rate 71.43%) and L. fermentum (3.97 log10 CFU/g (wet), detection rate 52.38%) are two dominant species of Lactobacillus. Study on Chinese child intestinal flora, especially on the compositions and numbers of key probiotics in the feces will be very helpful to the development of effective probiotics in future.