Comparison of the Real-Time PCR Tests for Factor V G1691A and Prothrombin G20210A with PCRRestriction Fragment Length Polymorphism and Direct Sequencing Tests

ABSTRACT We studied evidence of Bartonella henselae andBartonella clarridgeiae infection in 54 cats living in Jakarta, Indonesia. By using an indirect immunofluorescence assay, we found immunoglobulin G antibody to B. henselae in 40 of 74 cats (54%). The blood of 14 feral cats was cultured on rabbit blood agar plates for 28 days. Bartonella-like colonies were identified as B. henselae or B. clarridgeiae by using restriction fragment length polymorphism analysis and direct sequencing of the PCR amplicons. Of the cats sampled in the study, 6 of 14 (43%; all feral) were culture positive for B. henselae; 3 of 14 (21%; 2 feral and 1 pet) culture positive for B. clarridgeiae. This is the first report that documents B. henselae andB. clarridgeiae infections in Indonesian cats.

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Skin Pigmentation Differences between Mongolian, Korean, and Uzbekistan Ancient Human DNA Samples

BioMed Research International ◽

10.1155/2020/2585324 ◽

2020 ◽

Vol 2020 ◽

pp. 1-7

Author(s):

Munkhtsetseg Bazarragchaa ◽

Udval Uuganbayar ◽

Kwang-Ho Lee ◽

Kyung-Yong Kim ◽

Kijeong Kim

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Direct Sequencing ◽

Skin Pigmentation ◽

Melting Curve ◽

Melting Peak ◽

Degraded Dna ◽

Melting Curve Analysis ◽

Neolithic Period ◽

The Real

Background. This study reports the use of real-time PCR to identify the SNP rs1545397 in the intron region on the OCA2 gene from ancient and degraded DNA isolated from ancient human bones from Mongolia, Korea, and Uzbekistan. This SNP is a marker for skin pigmentation. LightCycler-based probes (HybProbes) were designed. A LightCycler (version 2.0) system was used for the real-time PCR. Results. The results of the real-time PCRs of three different genotypes of SNP rs1545397 were compared with those of the direct sequencing. Melting curve analysis was used for genotype determination. Three genotypes were distinguished: the homozygous T (T/T) SNP type formed a distinct melting peak at 53.3±0.14°C, the homozygous A (A/A) SNP type formed a distinct melting peak at 57.8±0.12°C, and the heterozygous A/T SNP type formed two distinct melting peaks at 53.3±0.17°C and 57.8±0.15°C. Mongolian aDNA samples tested in this study carried all three types of the SNP (A/T, A/A, and T/T) with no distinctly predominant type observed. In contrast, Korean aDNA samples carried the Asian genotype (T/T), while the Uzbekistan aDNA samples carried the European genotype (A/A) more often than the Asian genotype (T/T). Conclusions. Human Mongolian aDNA samples had A/T, A/A, and T/T SNP rs1545397 with no distinct predominant genotype. When combined with the archeological and aDNA studies of other coupling morphologies with aDNA, our results infer that Mongolia’s prehistoric population had considerable heterogeneity of skin color and morphological traits and that in the Neolithic period, a Eurasian or mixed population inhabited the western part of Mongolia.

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Lactobacillus Strain Diversity Based on Partial hsp60 Gene Sequences and Design of PCR-Restriction Fragment Length Polymorphism Assays for Species Identification and Differentiation

Applied and Environmental Microbiology ◽

10.1128/aem.01711-07 ◽

2007 ◽

Vol 74 (1) ◽

pp. 208-215 ◽

Cited By ~ 59

Author(s):

Giuseppe Blaiotta ◽

Vincenzina Fusco ◽

Danilo Ercolini ◽

Maria Aponte ◽

Olimpia Pepe ◽

...

Keyword(s):

Restriction Fragment Length Polymorphism ◽

16S Rrna ◽

Restriction Fragment ◽

Length Polymorphism ◽

Fragment Length ◽

Fragment Length Polymorphism ◽

Direct Sequencing ◽

Nucleotide Sequences ◽

Gene Sequences ◽

Restriction Fragment Length

ABSTRACT A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.

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