The next-generation high-throughput sequencing techniques have introduced a new way to assess the gut’s microbial diversity on the basis of 16S rRNA gene-based microbiota analysis. However, the precise appraisal of the biodiversity of Bifidobacterium species within the gut remains a challenging task because of the limited resolving power of the 16S rRNA gene in different species. The groEL gene, a protein-coding gene, evolves quickly and thus is useful for differentiating bifidobacteria. Here, we designed a Bifidobacterium-specific primer pair which targets a hypervariable sequence region within the groEL gene that is suitable for precise taxonomic identification and detection of all recognized species of the genus Bifidobacterium so far. The results showed that the novel designed primer set can specifically differentiate Bifidobacterium species from non-bifidobacteria, and as low as 104 cells of Bifidobacterium species can be detected using the novel designed primer set on the basis of Illumina Miseq high-throughput sequencing. We also developed a novel protocol to assess the diversity of Bifidobacterium species in both human and rat feces through high-throughput sequencing technologies using groEL gene as a discriminative marker.

BackgroundDyslipidemia is a well-established risk factor for cardiovascular disease. Serum lipids were affected by several gene polymorphisms, folate, homocysteine and other metabolite levels. We aim to investigate the effects of homocysteine metabolism enzyme polymorphisms (MTHTR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G) and their interactions with folate, homocysteine on serum lipid levels in Chinese patients with hypertension.MethodsParticipants were 480 hypertensive adults that enrolled in September to December 2005 from six different Chinese hospitals (Harbin, Shanghai, Shenyang, Beijing, Xi’an, and Nanjing). Known MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G genotypes were determined by PCR-RFLP methods. Serum folate was measured by chemiluminescent immunoassay, homocysteine was measured by high-performance liquid chromatography, serum lipids parameters were determined by an automatic biochemistry analyzer, low-density lipoprotein was calculated by Friedewald’s equation. Unitary linear regression model was used to assess the associations of gene polymorphisms, folate and homocysteine on serum lipid profiles. Unconditional logistic regression model was applied to test the interactions of folate, homocysteine and gene polymorphisms on dyslipidemia.ResultsNo correlations between gene polymorphisms and homocysteine on serum lipid profiles. Compared with normal folate patients, patients with low folate showed higher odds of hypertriglyceridemia (OR = 2.02, 95 % CI: 1.25-3.25, P = 0.004) and low levels of high-density lipoprotein cholesterol (OR = 1.88, 95 % CI: 1.07-3.28, P = 0.027). Each of four gene polymorphisms (MTHTR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G) combined with low folate showed higher odds of hypertriglyceridemia (P for trend: 0.049, 0.004, 0.007 and 0.005, respectively). MTHFR C677T and A1298C with low folate showed higher odds of low levels of high-density lipoprotein cholesterol (P for trend: 0.008 and 0.031).ConclusionsLow folate status and homocysteine metabolism gene polymorphisms (MTHTR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G) may have a synergistic effect increased the incidence of dyslipidemia in Chinese hypertensive population.

Both MTHFR and MTRR gene polymorphisms could be important genetic determinants of serum lipid levels in Chinese patients with hypertension. These findings need to be replicated in a larger sample.

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