Real-time Polymerase Chain Reaction Protocol Research Articles

Development of a real-time PCR protocol for the detection of chicken DNA in meat products

Food falsification is a pressing issue in today’s food industry, with fraudulent substitution of costly ingredients with cheaper alternatives occurring globally. Consequently, developing straightforward and efficient diagnostic systems to detect such fraud is a top priority in scientific research. The aim of the work was to develop a test system and protocol for polymerase chain reaction (PCR) to detect in food products of animal origin the substitution of expensive meat raw materials for by-products of poultry processing. For this, real-time polymerase chain reaction (RT-PCR) was used, which allows determining the qualitative and quantitative substitution in raw and technologically prepared products. Other methods for detecting falsification – enzyme immunoassay (ELISA/ELISA) or express methods in the form of a lateral flow immunoassay are less informative. The extraction of nucleic acids for real-time polymerase chain reaction depends on the source matrix, with higher concentrations obtained from germ cells and parenchymal organs. Extraction from muscle and plant tissues is more challenging, but thorough grinding of these samples improves nucleic acid concentration by 1.5 times using DNA extraction kits. The selection of primers and fluorescent probes through GenBank and PCR Primer Design/DNASTAR software enables efficient amplification and identification of target chicken DNA fragments in various matrices.

Detection of pathogenic Vibrio spp. in foods: polymerase chain reaction-based screening strategy to rapidly detect pathogenic Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus in bivalve mollusks and preliminary results.

The majority of human diseases attributed to seafood are caused by Vibrio spp., and the most commonly reported species are Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae. The conventional methods for the detection of Vibrio species involve the use of selective media, which are inexpensive and simple but time-consuming. The present work aimed to develop a rapid method based on the use of multiplex real-time polymerase chain reaction (PCR) to detect V. parahaemolyticus, V. vulnificus, and V. cholerae in bivalve mollusks. 30 aliquots of bivalve mollusks (Mytilus galloprovincialis) were experimentally inoculated with two levels of V. parahaemolyticus, V. vulnificus, and V. cholerae. ISO 21872-1:2017 was used in parallel for qualitative analysis. The limit of detection of 50% was 7.67 CFU/g for V. cholerae, 0.024 CFU/g for V. vulnificus, and 1.36 CFU/g for V. parahaemolyticus. For V. vulnificus and V. cholerae, the real-time PCR protocol was demonstrated to amplify the pathogens in samples seeded with the lowest and highest levels. The molecular method evaluated showed a concordance rate of 100% with the reference microbiological method. V. parahaemolyticus was never detected in samples contaminated with the lowest level, and it was detected in 14 samples (93.33%) seeded with the highest concentration. In conclusion, the developed multiplex real-time PCR proved to be reliable for V. vulnificus and V. cholerae. Results for V. parahaemolyticus are promising, but further analysis is needed. The proposed method could represent a quick monitoring tool and, if used, would allow the implementation of food safety.

Comparative performance of microscopy, nested PCR, and real-time PCR for screening avian haemosporidian parasites in Afrotropical starlings (family Sturnidae).

Prevalence studies of avian haemosporidian parasites frequently use microscopy and the nested polymerase chain reaction (PCR) protocols for detecting infections. Newer PCR protocols to detect parasites are being developed, with the distinct advantage of reducing screening cost and time, as well as increasing efficiency and sensitivity. The detection ability of microscopy and nested PCR was compared against a real-time PCR (qPCR) protocol using genomic DNA extracted from 240 bird blood samples collected from three starling species (Cape Starling, the Greater Blue-eared Starling, and the Wattled Starling; family Sturnidae) in the Kruger national park, South Africa. All three protocols successfully detected avian haemosporidian parasites with the qPCR having a considerable edge against the other two methods. Fifteen unique cytochrome b lineages were identified of which seven were new lineages. Microscopy and nested PCR recorded similar prevalence (32.92% and 35.42% respectively). The qPCR protocol used here, although more sensitive (52.92% prevalence), is not able to differentiate between parasite genera but provides the opportunity to screen a large number of samples in large-scale studies within a specific region. This study recommends the development and adoption of new molecular protocols with increased sensitivity and accuracy in prevalence studies. Nevertheless, microscopy remains essential for the morphological description of parasites and for distinguishing between abortive and successful chronic infections. The PCR-based method displays the detection of the parasitic genome but does not reveal whether parasites have or will develop into a successful infection.

Epithelial-to-Mesenchymal Transition Gene Signature in Circulating Melanoma Cells: Biological and Clinical Relevance.

The most promising method for monitoring patients with minimal morbidity is the detection of circulating melanoma cells (CMCs). We have shown that CD45-CD146+ABCB5+ CMCs identify a rare primitive stem/mesenchymal CMCs population associated with disease progression. The epithelial-to-mesenchymal transition (EMT) confers cancer cells a hybrid epithelial/mesenchymal phenotype promoting metastatization. Thus, we investigated the potential clinical value of the EMT gene signature of these primitive CMCs. A reliable quantitative real-time polymerase chain reaction (qRT-PCR) protocol was settled up using tumor cell lines RNA dilutions. Afterwards, immune-magnetically isolated CMCs from advanced melanoma patients, at onset and at the first checkpoint (following immune or targeted therapy), were tested for the level of EMT hallmarks and EMT transcription factor genes. Despite the small cohort of patients, we obtained promising results. Indeed, we observed a deep gene rewiring of the EMT investigated genes: in particular we found that the EMT gene signature of isolated CMCs correlated with patients' clinical outcomes. In conclusion, We established a reliable qRT-PCR protocol with high sensitivity and specificity to characterize the gene expression of isolated CMCs. To our knowledge, this is the first evidence demonstrating the impact of immune or targeted therapies on EMT hallmark gene expressions in CMCs from advanced melanoma patients.

Newcastle Disease Virus in Poultry: Current Status and Control Prospects

Since its first appearance in Java Island, Indonesia, in 1926, Newcastle disease has significantly impacted the global poultry industry, leading to substantial economic losses. The disease has rapidly spread worldwide, becoming endemic in many countries where agriculture is the primary source of national income. The present study aimed to present a comprehensive review of the recent literature on the Newcastle disease virus to contribute to understanding the virus and its control measures in poultry and provide an updated perspective on current knowledge. All strains of the Newcastle disease virus are classified under a single serotype; however, they are grouped into two classes and have been found to have emerging genetic diversity. Although various molecular diagnostic protocols have been developed, many have limitations. Nucleotide variability in the F gene of the Newcastle disease virus seems to explain the false-negative results provided by different real-time reverse transcription polymerase chain reaction protocols. Vaccination combined with biosecurity measures has been shown to limit the devastating effect of the Newcastle disease virus. However, the current vaccines are not effective enough to prevent viral shedding and infection of vaccinated animals. The efficacy of the vaccine strains utilized for decades is being scrutinized, raising questions about their effectiveness over time. The development of reverse genetics offers promising prospects for exploring new generations of attenuated vaccines capable of protecting poultry against clinical diseases and infections, such as Newcastle disease.

Evaluation of the Small Heat Shock Protein Family Members HSPB2 and HSPB3 in Bladder Cancer Prognosis and Progression.

Bladder cancer (BlCa) represents the sixth most commonly diagnosed type of male malignancy. Due to the clinical heterogeneity of BlCa, novel markers would optimize treatment efficacy and improve prognosis. The small heat shock proteins (sHSP) family is one of the major groups of molecular chaperones responsible for the maintenance of proteome functionality and stability. However, the role of sHSPs in BlCa remains largely unknown. The present study aimed to examine the association between HSPB2 and HSPB3 expression and BlCa progression in patients, and to investigate their role in BlCa cells. For this purpose, a series of experiments including reverse transcription-quantitative PCR, Western blotting, MTT assay and flow cytometry were performed. Initial analyses revealed increased vs. human transitional carcinoma cells, expression levels of the HSPB2 and HSPB3 genes and proteins in high grade BlCa cell lines. Therefore, we then evaluated the clinical significance of the HSPB2 and HSPB3 genes expression levels in bladder tumor samples and matched adjusted normal bladder specimens. Total RNA from 100 bladder tumor samples and 49 paired non-cancerous bladder specimens were isolated, and an accurate SYBR-Green based real-time quantitative polymerase chain reaction (qPCR) protocol was developed to quantify HSPB2 and HSPB3 mRNA levels in the two cohorts of specimens. A significant downregulation of the HSPB2 and HSPB3 genes expression was observed in bladder tumors as compared to matched normal urothelium; yet, increased HSPB2 and HSPB3 levels were noted in muscle-invasive (T2-T4) vs. superficial tumors (TaT1), as well as in high-grade vs. low-grade tumors. Survival analyses highlighted the significantly higher risk for post-treatment disease relapse in TaT1 patients poorly expressing HSPB2 and HSPB3 genes; this effect tended to be inverted in advanced disease stages (muscle-invasive tumors) indicating the biphasic impact of HSPB2, HSPB3 genes in BlCa progression. The pro-survival role of HSPB2 and HSPB3 in advanced tumor cells was also evident by our finding that HSPB2, HSPB3 genes expression silencing in high grade BlCa cells enhanced doxorubicin toxicity. These findings indicate that the HSPB2, HSPB3 chaperone genes have a likely pro-survival role in advanced BlCa; thus, they can be targeted as novel molecular markers to optimize treatment efficacy in BlCa and to limit unnecessary interventions.

Evaluation of the cytokines response in buffaloes focused on its milk as a newly emerging indicator tracing for toxocariasis.

This study aimed to evaluate the immune responses and oxidative stress provoked by Toxocara vitulorum infection in buffaloes with special reference to milk parameters as an emerging tool. The use of the milk tool was reported for the first time in tracing T. vitulorum infection in Egyptian buffaloes. Intestine, milk, serum, and liver samples were gathered from flocks in Cairo and Giza districts to evaluate buffalo immune responses provoked by T. vitulorum. The compositional items and somatic cells of milk were monitored. The intestine and milk were evaluated for interleukin IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) by quantitative real-time polymerase chain reaction protocol and the analysis of malondialdehyde (MDA) as an oxidative stress marker. The mean percentages for the total solids, fats, proteins, lactose, salts, pH, and somatic cell count/ml in positive samples were 11.23±0.37, 5.1±0.17, 4.44±0.14, 3.9±0.14, 0.81±0.02, 6.8±0.22, and 4.23×106± 1.41×105cells/ml, respectively. A significant difference (p<0.05) was observed in the mean values of compositional items except for the total protein %, salts %, and pH. For T. vitulorum-contaminated samples, the milk IL-1β, IL-6, TNF-α, and MDA (nmol/ml) were 7±0.23, 18±0.6, 17±0.56, and 3.7±0.12, respectively (which were less than the values for intestinal cytokines). There is a statistical difference (p<0.05) between positive and negative samples in the intestinal, milk cytokines, and MDA. This study is an initial investigation of the utilization of intestine and milk cytokines in the evaluation of buffalo toxocariasis.

Her2/EGFR-PDGFR pathway aberrations associated with tamoxifen response in metastatic breast cancer patients

BackgroundMetastatic breast cancer (MBC) is a major health problem worldwide. Some patients improve on tamoxifen and others do not respond to treatment. Therefore, the aim of the current study is to assess genetic aberrations in the Her2/EGFR-PDGFR pathway associated with tamoxifen response in MBC patients.MethodsThis is a retrospective cohort study, including 157 hormone receptors positive, locally recurrent inoperable and/or MBC patients on tamoxifen treatment. Patients were categorized into 78 (49.7%) tamoxifen responders and 79 (50.3%) tamoxifen non-responder patients. Genetic aberrations of 84 genes involved in the Her2/EGFR-PDGFR pathway were assessed in the tumor tissue samples obtained from the patients using SA-Bioscience assay. The identified panel was correlated to patients’ response to treatment, to detect the differentially expressed genes in tamoxifen responders and non-responders.ResultsOne hundred twenty-three (78.3%) patients were estrogen receptor (ER) and progesterone receptor (PR) positive, 108 (68.8%) were ER only positive, and 78 (49.7%) were PR only positive. There were 56 genes overexpressed in the refractory group compared to responders. However, only five out of these 56 genes, Janus kinase 1 (JAK1), collagen type I alpha 1 (COL1A1), GRB2-associated binding protein 1 (GAB1), fibronectin-1 (FN1), and MAP kinase-interacting serine/threonine-protein kinase (MKNK1), showed statistical significance between the two groups. Patients with bone metastasis showed a better response to treatment compared to those with metastatic deposits in other sites such as visceral metastasis (P < 0.005).ConclusionsGenetic profiling using simple quantitative real-time polymerase chain reaction (qRT-PCR) protocols could be used to assess response to tamoxifen treatment in MBC patients. According to our data, a five-gene panel in the EGFR pathway (JAK1, COL1A1, GAB1, FN1 and MKNK1) could be used to categorize MBC patients into groups according to treatment response.

Prevalence of H63D and C282Y mutations in hereditary hemochromatosis (HFE) gene in Tripoli region of Libya

Background and Aims: Hereditary hemochromatosis (HH) is an autosomal recessive disorder, characterized by increased intestinal absorption of iron. Excessive amount of iron accumulates in the liver, pancreas, and heart, etc., and eventually leading to organ failure due to iron toxicity and death if untreated. The most common causes of HH are the C282Y and H63D mutations in HFE gene. This study aimed to identify the prevalence of H63D and C282Y alleles among the Libyan population in Tripoli region and to compare the results with other published data. Materials and Methods: This study included 300 randomly selected unrelated Libyan male blood donors, aged between 18 and 50 years. In-house hydrolysis probe real-time polymerase chain reaction and high-resolution melting analysis protocols were developed and employed as screening tools for H63D and C282Y genotyping, respectively, and direct DNA sequencing was used to confirm the results. Results: Seven subjects (2.33%) were detected as homozygous H63D mutation and 72 (24%) were detected as heterozygous, and only one subject was detected as a heterozygous C282Y mutant (0.33%) and no homozygous C282Y mutation was detected. Conclusion: In Libyans residing in Tripoli region, the allele frequency of C282Y was very rare and allele frequency of H63D was common.

First molecular detection and identification of Leishmania species in small wild rodents from Turkey.

Leishmaniasis is a parasitic disease infecting animals and humans. Two clinical forms (Visceral and cutaneous leishmaniasis) and four species are reported to be present in Turkey. Several studies have investigated canine and human leishmaniasis in Turkey but no study was performed to screen the infection among wild rodents, so far. The present study aims to investigate the role of small wild rodents as reservoir animals for Leishmania spp. in different regions of Turkey. Formalin-preserved tissue samples (spleen, liver, lung) of 712 rodents from 30 provinces were screened for the presence of Leishmania spp. DNA. Before DNA extraction, tissues were dried, rehydrated, and homogenated. Leishmania screening in rodent tissues and species determination was performed with a combination of real-time kDNA and ITS1 polymerase chain reaction protocols. Eight (1.12%) out of 712 animals were found to be positive for Leishmania spp. DNA and species typing revealed five L. infantum, two L. tropica and one L. major among positives. Leishmania major and L. infantum DNA were detected in Apodemus spp. from Zonguldak province located in the Western Black Sea Region, while L. tropica DNA was found in Meriones sp. and Gerbillus dasyurus from Adana and Hatay provinces located in Eastern Mediterranean Region of Turkey. The present study is first to report natural infection of L. infantum, L. major and L. tropica in small wild rodents in Turkey, suggesting their possible roles as reservoirs. Further studies are needed for planning epidemiological studies and also for developing rodent control measures in risky endemic areas to break the transmission cycle.

Prospective multicenter comparison of urine culture with PCR on dried blood spots using 2 different extraction and PCR methods in neonates suspected for congenital cytomegalovirus infection

To evaluate the potential of dried blood spots (DBS) as a congenital cytomegalovirus (cCMV) testing specimen, the laboratory diagnostic accuracy of polymerase chain reaction (PCR) on DBS was compared to viral urine cultures from neonates suspected for cCMV. Two different extraction methods (EasyMAG, bioMérieux versus Qiagen) and 2 real-time PCR protocols (in-house versus Argene) were compared. We were able to collect both DBS and urine samples in 6 Belgian neonatal units from 276 neonates suspected for cCMV registered in CMVREG (an online neonatal registry system). Forty-eight neonates (17.4%) were positive by viral culture in urine. Laboratory diagnostic accuracy parameters of DBS-PCR were both extraction method and PCR protocol dependent. Not all DBS-CMV-PCR methods successfully detected urine-culture–positive neonates born after first-trimester seroconversions. Interestingly, however, all urine-culture–positive neonates having clinical signs of cCMV did consistently score positive.

Urinary TMAO Levels Are Associated with the Taxonomic Composition of the Gut Microbiota and with the Choline TMA-Lyase Gene (cutC) Harbored by Enterobacteriaceae.

Gut microbiota metabolization of dietary choline may promote atherosclerosis through trimethylamine (TMA), which is rapidly absorbed and converted in the liver to proatherogenic trimethylamine-N-oxide (TMAO). The aim of this study was to verify whether TMAO urinary levels may be associated with the fecal relative abundance of specific bacterial taxa and the bacterial choline TMA-lyase gene cutC. The analysis of sequences available in GenBank grouped the cutC gene into two main clusters, cut-Dd and cut-Kp. A quantitative real-time polymerase chain reaction (qPCR) protocol was developed to quantify cutC and was used with DNA isolated from three fecal samples collected weekly over the course of three consecutive weeks from 16 healthy adults. The same DNA was used for 16S rRNA gene profiling. Concomitantly, urine was used to quantify TMAO by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). All samples were positive for cutC and TMAO. Correlation analysis showed that the cut-Kp gene cluster was significantly associated with Enterobacteriaceae. Linear mixed models revealed that urinary TMAO levels may be predicted by fecal cut-Kp and by 23 operational taxonomic units (OTUs). Most of the OTUs significantly associated with TMAO were also significantly associated with cut-Kp, confirming the possible relationship between these two factors. In conclusion, this preliminary method-development study suggests the existence of a relationship between TMAO excreted in urine, specific fecal bacterial OTUs, and a cutC subgroup ascribable to the choline-TMA conversion enzymes of Enterobacteriaceae.

Effects of SR-BI rs5888 and rs4238001 variations on hypertension

Abstract Background Scavenger receptor class B, type I (SR-BI), involved in reverse cholesterol pathway, is a multilipoprotein receptor and capable of binding HDL, LDL and VLDL. SR-BI may contribute to the development of hypertension due to accumulation of cholesterol in the vessel wall via transporting lipoproteins. Therefore, it was aimed to investigate the relationship between SR-BI rs5888 and rs4238001 variants in the patient with hypertension. Materials and methods Seventy three subjects diagnosed with hypertension and 76 healthy subjects constituted the patient and control group, respectively. Genomic DNA was isolated from peripheral blood samples and a real-time quantitative polymerase chain reaction protocol was performed to detect variations of rs5888 and rs4238001. The results were analyzed with the SPSS 22 program and p &lt; 0.05 was considered statistically significant. Results and discussion SR-BI rs4238001 variation did not show significant difference between patient and control group (p &gt; 0.05). In the SR-BI rs5888 variation; normal homozygous CC and heterozygous CT carriers had an average 2-fold lower risk of hypertension than those carrying the TT genotype (p &lt; 0.05). Conclusion SR-BI rs5888 TT variant may increase hypertension risk by reducing lipid transport to the liver from the vessel wall.

DEVELOPMENT OF A REAL-TIME POLYMERASE CHAIN REACTION PROTOCOL FOR DIAGNOSIS OF PASTEURELLOSIS

Pasteurellosis is a zoonotic disease of vertebrate animals, for which the etiological agent is Pasteurella multocida. The continuous high prevalence of P. multocida and regular periodic outbreaks of pasteurellosis in both wild and domestic animals, especially in the wild saiga populations in Kazakhstan, underscores the importance of developing a local molecular diagnostic system. Accordingly, the goal of the present study was to develop a real-time polymerase chain reaction (PCR) protocol to identify all serovars of P. multocida. The envC, ruvB, and QseC genes were chosen as targets, and real-time PCR protocols were developed using three pairs of primers and corresponding fluorescent probes characterized by high efficiency, sensitivity, and specificity. The sensitivity of the developed protocols to the sequences of the envC and QseC genes was 2.38 fg, and that to the ruvB gene was 76 pg. The high specificity of the PCR protocol was confirmed with a collection of samples from 92 species of bacteria and three species of higher eukaryotic organisms. The commercialization of these developed protocols will allow for the production of highly effective PCR test systems, and improve the diagnosis and treatment of pasteurellosis.

Pediatric HIV Infection and Decreased Prevalence of OPV Point Mutations Linked to Vaccine-associated Paralytic Poliomyelitis.

BackgroundMutations associated with prolonged replication of the attenuated polioviruses found in oral poliovirus vaccine (OPV) can lead to vaccine-derived poliovirus (VDPV) and cause paralysis indistinguishable from that caused by wild poliovirus. In response, the World Health Organization has initiated the transition to exclusive use of inactivated poliovirus vaccine (IPV), with OPV administration in cases of outbreak. However, it is currently unclear how IPV-only vaccination, well known to provide humoral but not mucosal immunity, will impact the development of paralysis causing OPV variants. Children infected with human immunodeficiency virus (HIV) have been documented to show decreased mucosal immunity following OPV vaccination. Thus, HIV-infected children vaccinated with OPV may serve as proxy for children with IPV-only vaccination.MethodsWe conducted a prospective study of Zimbabwean infants receiving OPV as part of their routine vaccination schedule. Stool samples collected from OPV-vaccinated children serially until age 24 months were tested for OPV serotypes using a real-time polymerase chain reaction protocol that quantifies the amount of mutant OPV variants found in each sample.ResultsOut of 2130 stool samples collected from 402 infants 365 stool samples were OPV positive: 313 from 212 HIV-noninfected (HIV−) infants and 52 from 34 HIV-infected (HIV+) infants. HIV− infants showed significantly higher proportions of OPV mutants when compared to HIV+ infants.ConclusionsHIV infection is associated with a reduced proportion of OPV vaccine associated paralytic polio mutants. These results suggest that OPV administered to individuals previously vaccinated only with IPV will show decreased propensity for OPV mutations.

Detection of foot-and-mouth disease virus in milk samples by real-time reverse transcription polymerase chain reaction: Optimisation and evaluation of a high-throughput screening method with potential for disease surveillance

This study aimed to evaluate the utility of milk as a non-invasive sample type for the surveillance of foot-and-mouth disease (FMD), a highly contagious viral disease of cloven-hooved animals. Four milking Jersey cows were infected via direct-contact with two non-milking Jersey cows that had been previously inoculated with FMD virus (FMDV: isolate O/UKG/34/2001). Milk and blood were collected throughout the course of infection to compare two high-throughput real-time reverse transcription polymerase chain reaction (rRT-PCR) protocols with different RT-PCR chemistries. Using both methods, FMDV was detected in milk by rRT-PCR one to two days before the presentation of characteristic foot lesions, similar to detection by virus isolation. Furthermore, rRT-PCR detection from milk was extended, up to 28 days post contact (dpc), compared to detection by virus isolation (up to 14 dpc). Additionally, the detection of FMDV in milk by rRT-PCR was possible for 18 days longer than detection by the same method in serum samples. FMDV was also detected with both rRT-PCR methods in milk samples collected during the UK 2007 outbreak. Dilution studies were undertaken using milk from the field and experimentally-infected animals, where for one sample it was possible to detect FMDV at 10−7. Based on the peak CT values detected in this study, these findings indicate that it could be possible to identify one acutely-infected milking cow in a typical-sized dairy herd (100–1000 individuals) using milk from bulk tanks or milk tankers. These results motivate further studies using milk in FMD-endemic countries for FMD surveillance.

Expression Analysis of miR-29b in Malignant and Benign Breast Tumors: A Promising Prognostic Biomarker for Invasive Ductal Carcinoma With a Possible Histotype-Related Expression Status

BackgroundAberrations in microRNA levels seem to provide valuable information regarding breast cancer prognosis and therapy. In this study, we sought to analyze miR-29b expression in breast tumors and thus explore its clinical value. Materials and MethodsOne hundred twenty-one malignant and 56 benign breast tissue specimens were collected and subjected to extraction of total RNA, which was polyadenylated and reverse transcribed to cDNA. Subsequently, a highly sensitive quantitative real-time polymerase chain reaction protocol was developed and miR-29b levels, estimated via the comparative CT method, were finally subjected to comprehensive statistical analysis. ResultsMiR-29b levels did not differ between the analyzed benign and malignant breast tissue specimens, but were found to be significantly (P = .010) decreased in invasive ductal adenocarcinomas compared with their lobular counterparts, albeit receiver operating characteristics curve analysis did not verify the latter correlation. Additionally, miR-29b expression was elevated in samples with positive estrogen receptor status (P = .021) in the overall population, whereas it was negatively correlated (P = .035) with primary tumor staging in the ductal subset and increased in poorly-differentiated tumors of lobular origin (P = .041). Furthermore, Kaplan–Meier and Cox regression analyses showed that patients with ductal carcinoma and elevated miR-29b levels had a significantly longer disease-free survival (P = .010) and a lower risk to relapse (hazard ratio = 0.35, 95% confidence interval, 0.15-0.81; P = .014). ConclusionOur results provide evidence that miR-29b levels constitute a promising biomarker of favorable prognosis for patients with invasive ductal breast carcinoma and imply that its expression status might be affected by the histological origin of breast malignancy.

Study the Cross-talk Between Hepatitis B Virus Infection and Interferon Regulatory Factors in Liver Transplant Patients

Background: Interferon regulatory factors (IRFs) as immunoregulatory molecules have a determinative antiviral role in liver transplantation outcomes and graft rejection. Hepatitis B virus (HBV) and its antigen derivatives also choose some strategies to escape from innate immune responses. Objectives: The current study aimed at evaluating inflammatory cross-talks between pattern recognition receptors (PRRs) signaling components such as IRF3 and IRF7 with HBV infection in mRNA levels in patients undergoing liver transplantation. Methods: The 46 HBV infected liver recipients were divided into rejection experienced (20) and not experienced (26) groups and a healthy control group was also considered. Peripheral mononuclear cells (PBMCs) were isolated form each studied patient on the days 1, 4, and 7 in post-transplant period. After RNA extraction and cDNA synthesis from each collected sample, the expression levels of IRF3 and IRF7 genes were evaluated using in-house SYBER Green based the real-time polymerase chain reaction (PCR) protocols. Results: The overexpression of mRNA levels of IRF3 (3.37 folds) and IRF7 (1.74 folds) on the day 1 were found in patients experiencing rejection, compared with non-rejected ones, based on initial ischemia/reperfusion (I/R) injuries. But, the mRNA levels of IRF3 (0.53 folds) and IRF7 (0.74 folds) on the day 4 were downregulated in patients with rejected transplantation, compared with non-rejected ones. Finally, reducing the expression of IRF3 (0.54 fold) on the day 7 and upregulation of IRF7 (2.38 fold) on the day 7 were found in rejected liver recipients, compared with non-rejected ones in post-transplant period. Conclusions: Downregulation of IRF3 expression in patients with HBV infection, who experienced rejection episodes in the first week post-liver transplantation indicated that they may be at higher risk for acute rejection; the hypothesis, which should be investigated in further studies.

Species-specific Real Time-PCR primers/probe systems to identify fish parasites of the genera Anisakis, Pseudoterranova and Hysterothylacium (Nematoda: Ascaridoidea)

Ascaridoid nematodes belonging to the genera Anisakis and Pseudoterranova are heteroxenous parasites, involving marine mammals as definitive hosts in their life-cycles, whereas crustaceans (krill), fish and squids acting as intermediate/paratenic hosts. These parasites are considered among the most important biological hazards present in “seafood” products. Indeed, larval stages of the Anisakis and Pseudoterranova have been reported as etiological agents of human infections (anisakidosis). We developed a primers/probe system for the identification of five species of anisakid nematodes belonging to the genera Anisakis (i.e. A. pegreffii and A. simplex (s. s.)), and Pseudoterranova (i.e. P. decipiens (s. s.), P. krabbei and P. bulbosa) to be used in a real time polymerase chain reaction (RT-PCR) with specific primers based on the mtDNA cox2 gene. Because those anisakid species could be also found in co-infection in some fish species with the raphidascarid nematode Hysterothylacium aduncum, a species-specific primer probe system to be used in RT-PCR for this nematode species was also developed.The detection limit and specificity of the primer/probe systems were evaluated for each of the six nematode species. Singleplex and multiplex RT-PCR protocols were defined and tested. The detection limit of the nematode species tissue was lower than 0.0006ng/μl. Efficiency (E) of primers/probe systems developed was carried out by standard curve; E value varied between 2.015 and 2.11, with respect to a perfect reaction efficiency value of E=2. Considering the sensibility and quantitative nature of the assays, the new primers/probe system may represent a useful tool for future basic and applied research that focuses on the identification of Anisakis spp., Pseudoterranova spp. and H. aduncum larvae in fish, even in co-infections, with a potential for application in fish farming, fish processing industries, fish markets, and food producers.

Impact of the BCR-ABL1 fusion transcripts on different responses to Imatinib and disease recurrence in Iranian patients with Chronic Myeloid Leukemia

BackgroundOne genomic breakpoint can result in variable BCR-ABL1 transcript types due to alternative splicing. The influence of different BCR-ABL1 transcript types on clinical outcome is still controversial. Aim of the studyThe objective of this analysis was to determine the impact of transcript type on response, clinical outcome, recurrence risk after treatment with Imatinib mesylate in Chronic Myeloid Leukemia (CML) patients. MethodsSixty CML patients in chronic phase were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and banding standard protocols. ResultsThere was a significant difference in collective incidence of complete cytogenetic response (CCR) between the e14a2 and e13a2 groups (P=0.04). The median time to achieve CCR was shorter in e14a2 patients than to e13a2 (P=0.01). This finding is paralleled by the molecular response where the median of the BCR-ABL1/ABL1 expression levels were significantly lower in e14a2 transcript compared to e13a2 type at 3, 6, 9 and 12months from the start of therapy (P<0.01). The probability of recurrence after treatment discontinuation was 9.33 fold higher in e13a2 transcript, that is reported here for the first time (χ2=5.49; P=0.01; OR: 9.33; 95% CI: 1.59, 54.67). No significant difference was observed regarding overall survival (OS), although Patients with e14a2 transcript displayed a significant tendency toward a higher event free survival (EFS) ratio (P=0.03). ConclusionWe found that patients with the e14a2 transcript achieved better and faster responses to Imatinib mesylate. In this study, parallel data regarding molecular and cytogenetic responses, impact of transcript type on the probability of recurrence might suggest a general outcome that the type of transcript can be used as a prognostic marker at diagnosis.