PCR-RFLP Approaches Research Articles

Evaluating the effectiveness of DNA-based methods to detect mislabelling and adulteration of chestnut honey

Honey is a naturally sweet and viscous substance that is consumed worldwide for its specific flavour, taste and biological properties, which in turn depend on the type of plant from which bees collect nectar. The quality of honey and, in particular, the information on the product label determines consumer choice and market price. In this study, the true botanical composition of chestnut monofloral honeys produced in Calabria (South Italy) was determined by DNA barcoding, PCR-RFLP and LAMP approaches, all based on the chloroplast trnL-trnF intergenic spacer region. All three DNA-based techniques were reliable in unambiguously identifying the floral composition of the different honeys tested at species level. However, the LAMP assay has the advantage over DNA barcoding and PCR-RFLP of greatly simplifying and speeding up the identification of Castanea sativa as the dominant plant species of the Calabrian chestnut honeys. This may encourage the adoption of this method by the food industry for the rapid detection of intentionally or accidentally mislabelled ingredients in this valuable natural product, thus ensuring consumer safety and protection.

Association of T2285C polymorphism in PARP1 gene coding region with its expression, activity and NSCLC risk along with prognosis.

Poly (ADP-ribose) polymerase-1 (PARP1), a DNA repair gene, is the crucial player in the maintenance of genome integrity. T2285C polymorphism in coding region of PARP1 has been reported to be associated with susceptibility to tumours. We explored the relationship and mechanism of T2285C polymorphism of PARP1 to its expression and activity along with risk and prognosis in non-small cell lung cancer (NSCLC). mRNA expression was measured using quantitative RT-PCR assay or collected from TCGA dataset. Protein expression was examined with immunoblotting assay. Genotypes were determined by PCR-RFLP and sequencing approaches. PARP1 activity was determined with enzyme activity assay. Regulation of SIRT7 to PARP1 was determined by overexpression and small interference experiment. Association of PARP1 T2285C polymorphism with NSCLC risk was evaluated via multiple logistic regression analysis. Comparison of treatment response and progression-free survival (PFS) of NSCLC patients among different genotypes or regimens was made by chi-square test. Results indicated that mRNA and protein expression of PARP1 dramatically increased in NSCLC tissues in comparison with paired para-carcinoma tissues (P < 0.05). TC/CC mutant genotypes were associated with markedly enhanced PARP1 mRNA level compared with TT genotype (P = 0.011). No significant difference was discovered in PARP1 protein expression among TT, TC or CC genotypes (P > 0.05). Subjects with variant allele C had higher risk of NSCLC in comparison with allele T carriers [odds ratio = 1.560; P = 0.000]. NSCLC patients carrying mutational TC or CC genotypes were correlated with unfavourable response to platinum-based chemotherapy (TT vs. TC vs. CC, P = 0.010), and shorter PFS compared with TT genotype (TT vs. TC vs. CC, P = 0.009). T2285C mutation of PARP1 resulted in the enhancement of its mRNA, but the decrease of enzyme activity in tumour cell. Overexpression of SIRT7 attenuated PARP1 expression and activity. These findings suggest the variant allele C of T2285C polymorphism of PARP1 linked to an increase of NSCLC risk, and unfavourable efficacy and prognosis of NSCLC patients with platinum-based chemotherapy, which might be associated with enhancement of its mRNA expression and the diminishment of activity. Identification of PARP1 T2285C polymorphism and mRNA expression may be the promising way for the individualised treatment of NSCLC.

A link between promoter polymorphisms of the transforming growth factor β1 (TGFB1) and TGF-β1 receptor II (TGFBR2) genes and relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is achronic progressive autoimmune disease characterised by nerve demyelination, mediated by myelin-specific Th1 autoreactive cells. Transforming growth factor1 (TGF-1) is aregulatory cytokine involved in MS aetiology by maintaining CD4+ cell differentiation and preventing autoimmune responses. Because of the important role of the TGF-1 signalling pathway in MS aetiopathogenesis, we aimed to investigate the association of two DNA polymorphisms: TGFB1C[-509]T and TGFBR2G[-875]Aand their combined genotypes with the risk of MS development in acohort of Bulgarian patients. The effect of the two promoter polymorphisms on the disease onset was also assessed. In the study, acohort of 183 patients with relapsing-remitting multiple sclerosis (RRMS) and 307 sex- and age-matched healthy subjects were recruited. Genotyping of the TGFB1C[-509]T (rs1800469) and TGFBR2G[-875]A(rs3087465) polymorphisms was performed by PCR-RFLP and PIRA-PCR approaches. Frequencies of the TGFB1T[-509]T genotype and TGFB1[-509]*T-allele were lower in RRMS men than in control healthy men (15.7% vs. 26.9%, 37.3% vs. 50.7%, respectively). Among males, the TGFB1T[-509]T genotype was related to asignificantly reduced risk of RRMS (OR = 0.360, 95% CI: 0.126-1.028, p = 0.05) in comparison to the TGFB1C[-509]C genotype. Also, TGFB1[-509]*T-allele was more common in men with RRMS than in healthy men relative to the TGFB1[-509]*C-allele and was associated with astatistically significant protective effect (OR = 0.576, 95% CI: 0.341-0.974, p = 0.039). The combination of TGFB1T[-509]T/TGFB1T[-509]C and TGFBR2G[-875]Agenotypes among men was associated with asignificant protective effect compared to the wild-type homozygous TGFB1C[-509]C and TGFBR2G[-875]G genotypes (OR = 0.268, 95% CI: 0.088-0.818, p = 0.018). No significant association between rs1800469 and rs3087465 was observed among females with and without (controls) RRMS. In summary, we suggest that in males, ahigher TGF-1 level determined by TGFB1T[-509]T genotype in combination with the TGFBR2G[-875]Agenotype might be aprotective factor against RRMS development.

Complex epidemiology and zoonotic potential for Cryptosporidium suis in rural Madagascar

Cryptosporidium spp. is the most important parasitic diarrheal agent in the world, is among the top four causes of moderate-to-severe diarrheal disease in young children in developing nations, and is problematic as an opportunistic co-infection with HIV. In addition, Cryptosporidium is a persistent challenge for livestock production. Despite its zoonotic potential, few studies have examined the ecology and epidemiology of this pathogen in rural systems characterized by high rates of overlap among humans, domesticated animals, and wildlife. To improve our understanding of the zoonotic potential of Cryptosporidium species in the rural tropics, we screened humans, livestock, peridomestic rodents, and wildlife using PCR-RFLP and sequencing-based approaches to distinguish species of Cryptosporidium in rural southeastern Madagascar. Cryptosporidium of multiple species/genotypes were apparent in this study system. Interestingly, C. suis was the dominant species of Cryptosporidium in the region, infecting humans (n=1), cattle (n=18), pigs (n=3), and rodents (n=1). The broad species range of C. suis and the lack of common cattle Cryptosporidium species (Cryptosporidium parvum and Cryptosporidium andersoni) in this system are unique. This report represents the fifth confirmed case of C. suis infection in humans, and the first case in Africa. Few rural human and livestock populations have been screened for Cryptosporidium using genus-specific genotyping methods. Consequently, C. suis may be more widespread in human and cattle populations than previously believed.

PCR-RFLP approaches to easily identify Pleuronectes platessa from other flatfishes: a rapid and efficient tool to control label information

European plaice (Pleuronectes platessa) is quite frequently substituted with other flatfish, especially with Yellow fin sole (Limanda aspera), which differs not only in meat quality but also particularly in its origin and manipulation chain. We propose an integrated approach of laboratory and in silico mtDNA PCR-RFLP procedures generating a set of restriction patterns easily resolvable in agarose gel, able to discriminate with certainty P. platessa from other 20 flatfish species. The herein proposed procedure is an economical and valid tool in detecting mislabelled seafoods.

Association of seven functional polymorphisms of one-carbon metabolic pathway with total plasma homocysteine levels and susceptibility to Parkinson's disease among South Indians

This study from South India was performed to ascertain the impact of seven functional polymorphisms of one-carbon metabolic pathway on total plasma homocysteine levels and susceptibility to PD. A total of 151 cases of Parkinson's disease and 416 healthy controls were analyzed for fasting plasma homocysteine levels by reverse phase HPLC. PCR-RFLP approaches were used to analyze glutamate carboxypeptidase II (GCPII) 1561 C>T, reduced folate carrier 1 (RFC1) 80 G>A, cytosolic serine hydroxymethyl transferase (cSHMT) 1420 C>T, methylene tetrahydrofolate reductase (MTHFR) 677 C>T, methionine synthase (MTR) 2756 A>G and methionine synthase reductase (MTRR) 66 A>G polymorphisms. PCR-AFLP was used for the analysis of thymidylate synthase (TYMS) 5′-UTR 28bp tandem repeat. PD cases exhibited elevated plasma homocysteine levels compared to controls (men: 28.8±6.9 vs. 16.4±8.8μmol/L; women: 25.4±5.3 vs. 11.2±5.1μmol/L). Homocysteine levels showed positive correlation with male gender (r=0.39, p<0.0001) and MTRR 66 A>G (r=0.31, p<0.0001) whereas an inverse correlation was observed with cSHMT 1420 C>T polymorphism. MTRR 66 A>G polymorphism showed independent risk for PD (OR: 3.42, 95% CI: 2.35–4.98) whereas cSHMT 1420 C>T conferred protection against PD (OR: 0.11, 95% CI: 0.07–0.17). Multifactor dimensionality reduction analysis showed synergistic interactions between MTHFR 677 C>T and MTRR 66 A>G, whereas cSHMT 1420 C>T exhibited counteracting interactions in altering susceptibility to PD. To conclude, PD cases exhibited hyperhomocysteinemia and MTRR 66 A>G and cSHMT 1420 C>T gene variants were shown to modulate PD risk by altering the homocysteine levels.

Investigation of genetic effect of growth hormone gene on antler production in Chinese Sika deer Cervus nippon

Antler is one of the most important products of Sika deer (Cervus nippon) and has great economic values. The genetic mechanisms and genetic markers associated with antler growth of Sika deer have been rarely reported. In the present study, the growth hormone (GH) gene was analyzed as a candidate gene for antler growth in Chinese Sika deer. Partial sequence of GH exon3 and 5 of Sika deer were obtained and four novel SNPs (ss325995760, ss325995761, ss325995762 and ss325995763) were then identified. Genotyping of four single nucleotide polymorphisms (SNPs) were implemented in both Shuangyang Sika deer (n = 69) and Dongfeng Sika deer (n = 90) using PCR-RFLP or DNA sequencing approaches, and association between different genotypes and anlter weight traits were analyzed. The SNP ss325995760 was significantly (P < 0.05) associated with average antler yield in Shuangyang Sika deer while it was not associated in Dongfeng Sika deer. However, other three SNPs were not associated with the traits. The preliminary result implied that the identified SNPs of GH gene could be considered one of the candidate markers for marker assisted selection in Sika deer but further studies are warranted. Key words: Sika deer, growth hormone, single nucleotide polymorphisms (SNPs)association, antler.

The capacity of Mycobacterium tuberculosis Complex Species and Mycobacterium bovis BCG Substrains Specific Identification – Implications for Optimized PCR-based Diagnostics in Adverse Events Following Vaccination Suspected Cases

The capacities of differentiation of Mycobacterium bovis BCG from other members of M. tuberculosis complex species using PCR-RFLP, multiplex PCR, and PCR-based genomic deletion analysis approaches were compared. In the study, mycobacteria isolated from patients suspected of adverse events following vaccination with BCG, primarily classified according presence of RD1 marker as virulent and avirulent mycobacteria, were used. The PCR-based genomic deletion analysis was found the best option for mycobacteria diagnostics improvement, as it was capable precisely differentiate virulent and avirulent mycobacteria or virulent species of M. tuberculosis complex. The routine confirmation of mycobacteria species in the cases of adverse events following BCG vaccination is highly expected, especially in clinical practice of patients with primary immunodeficiency.

Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) expression is epigenetically regulated by one-carbon metabolism in invasive duct cell carcinoma of breast

In view of recent studies highlighting the prognostic relevance of expression and CpG island methylator phenotype (CIMP) of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) in invasive duct cell carcinoma (IDC), we hypothesized in this article that impaired one-carbon metabolism might influence CIMP phenotype of BNIP3. In order to substantiate the prognostic relevance of BNIP3, we explored its association with 8-oxo-2'deoxyguanosine (8-oxodG), a marker of oxidative stress with prognostic relevance. BNIP3 expression and CIMP phenotype were studied using semi-quantitative RT-PCR and combined bisulfite restriction analysis (COBRA), respectively, in 56 IDC tumors. Eight polymorphisms in one-carbon metabolism were studied using PCR-RFLP and PCR-AFLP approaches. 8-oxodG was measured using competitive ELISA kit. BNIP3 was found to be upregulated in IDC (cases vs. controls: 0.94 ± 0.05 vs. 0.18 ± 0.08, P < 0.0001). COBRA analysis confirmed hypomethylation of BNIP3 promoter CpG island in these cases. CIMP phenotype of BNIP3 showed positive association with tubule formation (P = 0.034) and methionine synthase reductase (MTRR) A66G (P = 0.002); inverse association with cytosolic serine hydroxyl methyltransferase (cSHMT) C1420T (P < 0.005) and 8-oxodG (<10% vs. >10% methylation: 7.24 ± 2.77 ng/ml vs. 4.42 ± 2.93 ng/ml, P < 0.0005); and no association with nuclear pleomorphism or mitotic index or ER, PR, and HER statuses. Synergistic effect of MTR A2756G and MTRR A66G variants on BNIP3 hypermethylator phenotype was clearly evident (P < 0.0007). MTRR A66G and cSHMT C1420T polymorphisms influence CIMP phenotype of BNIP3, thus epigenetically regulating BNIP3 in breast cancer. The linear association between BNIP3 and 8-oxodG substantiates the role of BNIP3 as redox sensor as well as prognostic marker in breast cancer.

Comparative analyses reveal discrepancies among results of commonly used methods for Anopheles gambiae molecular form identification

BackgroundAnopheles gambiae M and S molecular forms, the major malaria vectors in the Afro-tropical region, are ongoing a process of ecological diversification and adaptive lineage splitting, which is affecting malaria transmission and vector control strategies in West Africa. These two incipient species are defined on the basis of single nucleotide differences in the IGS and ITS regions of multicopy rDNA located on the X-chromosome. A number of PCR and PCR-RFLP approaches based on form-specific SNPs in the IGS region are used for M and S identification. Moreover, a PCR-method to detect the M-specific insertion of a short interspersed transposable element (SINE200) has recently been introduced as an alternative identification approach. However, a large-scale comparative analysis of four widely used PCR or PCR-RFLP genotyping methods for M and S identification was never carried out to evaluate whether they could be used interchangeably, as commonly assumed.ResultsThe genotyping of more than 400 A. gambiae specimens from nine African countries, and the sequencing of the IGS-amplicon of 115 of them, highlighted discrepancies among results obtained by the different approaches due to different kinds of biases, which may result in an overestimation of MS putative hybrids, as follows: i) incorrect match of M and S specific primers used in the allele specific-PCR approach; ii) presence of polymorphisms in the recognition sequence of restriction enzymes used in the PCR-RFLP approaches; iii) incomplete cleavage during the restriction reactions; iv) presence of different copy numbers of M and S-specific IGS-arrays in single individuals in areas of secondary contact between the two forms.ConclusionsThe results reveal that the PCR and PCR-RFLP approaches most commonly utilized to identify A. gambiae M and S forms are not fully interchangeable as usually assumed, and highlight limits of the actual definition of the two molecular forms, which might not fully correspond to the two A. gambiae incipient species in their entire geographical range. These limits are discussed and operational suggestions on the choice of the most convenient method for large-scale M- and S-form identification are provided, also taking into consideration technical aspects related to the epidemiological characteristics of different study areas.

Bacterial diversity, organic pollutants and their metabolites in two aeration lagoons of common effluent treatment plant (CETP) during the degradation and detoxification of tannery wastewater

In this study, PCR-RFLP and GC-MS approaches were used to characterize the bacterial diversity, organic pollutants and metabolites during the tannery wastewater treatment process at common effluent treatment plant (CETP). Results revealed that the bacterial communities growing in aeration lagoon-I were dominated with Escherichia sp., Stenotrophomonas sp., Bacillus sp. and Cronobacter sp. while that of aeration lagoon-II prevailed with Stenotrophomonas sp., and Burkholderiales bacterium, respectively. The HPLC and GC-MS analysis revealed that most of the organic pollutants detected in untreated tannery wastewater samples were diminished from bacterial treated tannery wastewater samples. Only two pollutants i.e. L-(+)-lactic acid and acetic acid could not be degraded by bacteria whereas benzene and 2-hydroxy-3-methyl-butanoic acid was produced as new metabolites during the bacterial treatment of tannery wastewater in aeration lagoon II of CETP. Further, it was observed that after bacterial treatment, the toxicity of tannery effluent was reduced significantly allowing 90% seed germination.

DNA Repair Gene Polymorphisms Predict Favorable Clinical Outcome in Advanced Non–Small-Cell Lung Cancer

Background Genetic polymorphisms of genes involved in DNA repair and glutathione metabolic pathways may affect patients' response to platinum-based chemotherapy. We retrospectively assessed whether single nucleotide polymorphisms (SNP) of DNA-repair genes ERCC1, XPD, XRCC1 and glutathione S-transferase genes GSTP1, GSTT1 and GSTM1 predict overall survival (OS), response and toxicity in 119 non–small-cell lung cancer (NSCLC) patients treated with platinum-based regimens as first- or second-line chemotherapy. Patients and Methods Patients' genotypes were determined by PCR-RFLP and sequencing approaches. Results ERCC1 (Asn118Asn) genotype was significantly associated with response to treatment. Patients with either one or two C alleles (C/C, C/T) at Asn118Asn were more likely to respond to platinum-based chemotherapy compared with those without the C allele (Odds ratio, 0.10; 95% CI, 0.013-0.828; P = .033, by binary logistic regression). There was a significant association between the ERCC1 C8092A polymorphism and OS ( P = .009, by log-rank test), with median survival times of 9.8 (C/C) and 14.1 (C/A or A/A) months, respectively, suggesting that any copies of the A allele were associated with an improved outcome. Cox's multivariate analysis suggested that the joint effect of ERCC1 polymorphic variants (C8092A and N118N) (0 vs. 2, hazard ratio 2.5; 95% CI, 1.26–4.96; P = .009) as well as the XRCC1 N399Q polymorphism (AA vs. GA/GG, hazard ratio 3.1; 95% CI, 1.4–6.8; P = .005) were independent prognostic factors for OS in advanced NSCLC patients treated with platinum-based chemotherapy. Conclusion These findings support the notion that assessment of genetic variations of ERCC1 and XRCC1 could facilitate therapeutic decisions for individualized therapy in advanced NSCLC.

Simultaneous identification of molecular and mating types within the Cryptococcus species complex by PCR-RFLP analysis

The Cryptococcus species complex consists of two species, Cryptococcus neoformans and Cryptococcus gattii, which cause systemic infections in both immunocompromised and immunocompetent patients. Both species have a bipolar mating system, with mating type (MAT) alpha being predominant in clinical and environmental isolates. The strains of the Cryptococcus species complex have been divided into eight major molecular types, which show differences in epidemiology, biology and pathogenicity. In this study, two PCR-RFLP analyses, based on the CAP1 and GEF1 genes, which are both located at the MAT locus, were developed for simultaneous identification of the molecular and mating types of isolates of the Cryptococcus species complex. The molecular and mating types of all 144 cryptococcal isolates, including rare subtypes, were successfully determined by both PCR-RFLP approaches. Pattern analysis of the AD hybrids revealed that the serotype A MATa allele in strains of AaDalpha derived from genotype VNB, whereas the serotype A MATalpha allele among strains of AalphaDa and AalphaDalpha derived from molecular type VNI.

HLA-B*27 typing by PCR-restriction fragment length polymorphism.

In this study we describe the use of PCR-RFLP for genotyping HLA-B*27. A 557 bp fragment from HLA-B locus is amplified and subjected to digestion with StyI. The presence of B*27 is detected on electrophoresis by the appearance of 431 + 126 bp pattern. The same pattern could be obtained only for the very infrequent allele B*7301. However, this allele was not amplified in the B73 sample tested with the primers and conditions used in this study. Nevertheless, we have designed two PCR-RFLP approaches for separating these alleles. The PCR-RFLP method was tested on a panel of forty-three cell lines and applied to fifty spondyloarthritic patients and one-hundred-eighty healthy subjects. Given its robustness, technical simplicity and cost-effectiveness, we think that this method can be incorporated for routine use in most laboratories.

Genetic Characterization of the Asian Taenia, a Newly Described Taeniid Cestode of Humans

Recent studies on the epidemiologic pattern of taeniasis in Southeast Asia have indicated the existence of a third form of human Taenia, distinguishable from Taenia saginata and T. solium. Originally termed Taiwan Taenia, and first described in Taiwanese aboriginals, this newly recognized taeniid is now generally referred to as Asian Taenia since it has since been recorded in a number of other Asian countries. Here we have used a genetic yardstick approach to determine whether the Asian Taenia should most appropriately be considered as a new, distinct species or as a subspecies, strain, or variant of T. saginata, which previous studies have shown it closely resembles. Sequence variation in the 28S rRNA and mitochondrial cytochrome c oxidase I (COI) genes of a range of taeniid cestodes and the COI and rDNA internal transcribed spacer I polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) pattern differences in the Asian Taenia, T. saginata, and T. solium were used as markers of genetic identity. The PCR-RFLP approaches proved useful for rapid and unambiguous discrimination of Asian Taenia from the other two human species, whereas the mitochondrial and nuclear sequence comparisons indicate that the Asian Taenia is much more closely related to T. saginata than recognized taeniid species are to each other. The results support earlier conclusions that the Asian Taenia is a genetically distinct entity but is closely related to T. saginata, and suggest that its taxonomic classification as a subspecies or strain of T. saginata is more appropriate than formal designation as a new species. The very close relationship between Asian Taenia and T. saginata has public health implications in that the Asian form is unlikely to be an important cause of human cysticercosis because T. saginata cysticercosis, if it occurs at all in humans, is an extremely rare phenomenon.