METHODS: A total of 17 samples collected from December 2009 to January 2011 were analyzed. Reverse transcriptase polymerase chain reaction (PCR) was performed, followed by sequencing technique. Results were analyzed based on sequence information in GenBank. A second genotyping method (AmpliSens(®) HCV-1/2/3-FRT) was done, which differentiates HCV genotypes by means of real-time hybridization-fluorescence detection.
RESULTS: From 17 samples, four were untypeable by AmpliSens(®) HCV-1/2/3-FRT. Eleven of 13 (84.6%) results showed concordant genotypes. A specimen that was determined as genotype 3a by sequencing was genotype 1 by the AmpliSens(®) HCV-1/2/3-FRT. Another specimen that was genotype 1 by sequencing was identified as genotype 3 by AmpliSens(®) HCV-1/2/3-FRT.
CONCLUSION: HCV genotyping with AmpliSens(®) HCV-1/2/3-FRT using real-time PCR method provides a much simpler and more feasible workflow with shorter time compared to sequencing method. There was good concordance compared to sequencing method. However, more evaluation studies would be required to show statistical significance, and to troubleshoot discordant results. AmpliSens(®) HCV-1/2/3-FRT does differentiate between genotype but not until subtype level.
RESULTS: We used 43,310 SNPs to infer the population structure, evidence of local adaptation and sources of introduction. The overall genetic differentiation of this species was low. The native populations were differentiated into three genetic clusters, corresponding to the Yangtze, Pearl and Heilongjiang River Systems, respectively. The populations in Malaysia, India and Nepal were introduced from both the Yangtze and Pearl River Systems. Loci and genes involved in putative local selection for native locations were identified. Evidence of both positive and balancing selection was found in the introduced locations. Genes associated with loci under putative selection were involved in many biological functions. Outlier loci were grouped into clusters as genomic islands within some specific genomic regions, which likely agrees with the divergence hitchhiking scenario of divergence-with-gene-flow.
CONCLUSIONS: This study, for the first time, sheds novel insights on the population differentiation of the grass carp, genetics of its strong ability in adaption to diverse environments and sources of some introduced grass carp populations. Our data also suggests that the natural populations of the grass carp have been affected by the aquaculture besides neutral and adaptive forces.
METHODS: Three SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method.
RESULTS: The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed.
CONCLUSIONS: The KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.
METHODS AND RESULTS: Comparisons were made for Anyplex28 genotyping from 115 cervical samples extracted on the Hamilton, STARlet and the MP96. Two DNA concentrations were used for the MP96, one matched for sample input to the STARlet and another 5× concentration (laboratory standard). Agreement of HPV detection was 89·8% (κ = 0·798; P = 0·007), with HPV detected in 10 more samples for the MP96. There was a high concordance of detection for any oncogenic HPV genotype (κ = 0·77; P = 0·007) and for any low-risk HPV genotype (κ = 0·85; P = 0·008). DNA extracted at laboratory standard had a lower overall agreement 85·2% (κ = 0·708; P