Detection Of Genotypes Research Articles

Point‐of‐Care Diagnostic Platforms for Loop‐Mediated Isothermal Amplification

The loop‐mediated isothermal amplification (LAMP) method is one of the Nucleic acid amplification tests (NAATs) that allows for the amplification of target regions without using a thermal cycle. With its unique primer design, LAMP ensures the rapid replication of the targeted DNA region with high specificity and high efficiency. LAMP technology is used for diagnostic purposes in pathogen detection due to its ease of use, low cost, and simplicity without requiring complex equipment. A wide range of LAMP diagnostic platforms have been developed for applications in bacteria, virus, and parasitic pathogen detection. Herein, the methodology of LAMP technology and its applications in pathogen detection and SNP genotyping and mutation detection are discussed. Point‐of‐care (PoC) LAMP platforms designed with the principles of microfluidic chip technology, including LAMP‐on‐a‐chip, paper‐based LAMP, and smartphone‐based LAMP applications have been elaborated. LAMP technology represents a fast, robust, and reliable diagnostic platform for point‐of‐care testing.

Implications of Possible HBV-Driven Regulation of Gene Expression in Stem Cell-like Subpopulation of Huh-7 Hepatocellular Carcinoma Cell Line.

Elevated levels of STIM1, an endoplasmic reticulum Ca2+ sensor/buffering protein, appear to be correlated with poor cancer prognosis in which microRNAs are also known to play critical roles. The purpose of this study is to investigate possible HBV origins of specific microRNAs we identified in a stem cell-like subpopulation of Huh-7 hepatocellular carcinoma (HCC) cell lines with enhanced STIM1 and/or Orai1 expression that mimicked poor cancer prognosis. Computational strategies including phylogenetic analyses were performed on miRNome data we obtained from an EpCAM- and CD133-expressing Huh-7 HCC stem cell-like subpopulation with enhanced STIM1 and/or Orai1 expression originally cultured in the present work. Results revealed two putative regions in the HBV genome based on the apparent clustering pattern of stem loop sequences of microRNAs, including miR3653. Reciprocal analysis of these regions identified critical human genes, of which their transcripts are among the predicted targets of miR3653, which was increased significantly by STIM1 or Orai1 enhancement. Briefly, this study provides phylogenetic evidence for a possible HBV-driven epigenetic remodeling that alters the expression pattern of Ca2+ homeostasis-associated genes in STIM1- or Orai1 overexpressing liver cancer stem-like cells for a possible mutual survival outcome. A novel region on HBV-X protein may affect liver carcinogenesis in a genotype-dependent manner. Therefore, detection of the viral genotype would have a clinical impact on prognosis of HBV-induced liver cancers.

Morphological, Pomological, and Nutritional Value of Wild and Cultivated Rosehip (Rosa canina L.) Genotypes in Slavonia, Croatia

In this study, the morphological, pomological, and nutritional values of wild and cultivated rosehip fruits grown in the Slavonia region of eastern Croatia were studied. The results revealed significant differences in several morphological and pomological characteristics among the rosehip genotypes in terms of fruit weight, flesh weight, seed weight, and fruit flesh ratio, with no significant differences in fruit width, fruit length, fruit shape index, seed number per fruit, or seed length. The evaluated rosehip fruit genotypes differed significantly from each other in terms of hectoliter weight (kg), fruit bulk (cm3), and bulk density (kg/m3). For water–soluble extracts, ash, and pH, no statistical difference was found between naturally grown genotypes, but there was a significant difference between naturally grown and cultivated genotypes. Twenty-three major and trace elements were analyzed. The most abundant elements were K, Ca, Mg, and P in both cultivated and naturally grown fruits. The highest concentrations of microelements were Fe, Al, Mn, and Sr. The conventionally cultivated genotype L1 had the highest concentration of Fe and Na as essential elements for humans but also had the highest concentrations of Al, Sr, Ti, V, Cr, Pb, Co, Li, and As of all the genotypes studied. The naturally grown genotype L4 had the highest concentrations of S, Zn, Rb, and Cd and the lowest concentrations of Mg, K, and Ca among all studied genotypes. The data showed that the analyzed genotypes from eastern Croatia had good nutritional quality and variability, making them suitable as genetic resources and possibly leading to the detection of rosehip genotypes as potential sources of beneficial ingredients for human health.

Human papillomavirus prevalence, persistence and cervical dysplasia in females with cystic fibrosis

BackgroundA higher risk of human papillomavirus (HPV)-related cervical intra-epithelial neoplasia (CIN) is suspected among females with cystic fibrosis (CF). MethodsWe conducted a single center prospective cohort study among females attending the Lyon adult CF center. We performed a cervical cytology (Hologic Thinprep®) and HPV testing with genotyping (Clinical Arrays Papillomavirus; Genomica, enabling 35 genotype detection, 20 of which are high-risk (HR-HPV)) at inclusion. We followed all females with positive HPV tests at 6, 12 and 24 months to evaluate HPV persistence, and performed a colposcopy in cases of abnormal cytology. ResultsWe included eighty-five participants, 18 (21%) of whom were lung-transplanted. The mean age at inclusion was 31.9 (range 18-59) years. The prevalence of HPV (all types) was 31.8%. HR-HPV was found in 25.9% of the whole cohort, 44.4% of transplanted patients, and 20.1% of nontransplanted patients. Genotype-specific HR-HPV persistence at 12 months was 43.5% among transplanted and 34.6% among nontransplanted patients. Overall, 17.6% (15/85) of females had an abnormal cytology: 44.4% (8/18) among transplanted and 10.4% (7/67) among nontransplanted patients. CIN was identified in 12 (14.1%) patients (6 low-grade, 6 high-grade). High-grade CIN developed in 4 nontransplanted patients. ConclusionTransplanted females had high HR-HPV, abnormal cervical cytology and CIN prevalence rates compared to large published cohorts in the general non-CF population. Although HR-HPV prevalence and persistence were globally not significantly different in nontransplanted females compared to the general population, we reported high frequencies of abnormal cytology and CIN. Cervical cancer screening and prevention should be promoted among females with CF.

Prevalence and genetic diversity of Aeromonas veronii isolated from aquaculture systems in the Poyang Lake area, China.

The area around Poyang Lake is the main aquaculture area in Jiangxi Province, China, and an important base for the supply of freshwater aquatic products. Aquaculture in the Poyang Lake area is severely threatened by diseases caused by bacterial pathogens, and Aeromonas veronii has been the main pathogen in recent years. In this paper, ERIC-PCR genotyping, virulence gene and antimicrobial resistance gene detection, and drug susceptibility tests were carried out on 46 A. veronii isolates obtained from aquaculture systems in the Poyang Lake area from 2016 to 2020. The results showed that the A. veronii strains in the Poyang Lake area had high genetic diversity, and 46 strains produced 36 ERIC genotypes. There were no geographical and temporal differences in the cluster analysis results and no dominant clones. All 13 virulence genes tested were detected, and all isolates had harbored 2 or more virulence genes, with a maximum of 12 virulence genes detected. Among the 22 antimicrobial resistance genes selected, 15 were detected; 97.8% of the isolates contained 2 or more antimicrobial resistance genes, with a maximum of 9 antimicrobial resistance genes. Drug susceptibility tests showed that some strains were resistant to a variety of traditionally effective drugs for Aeromomas, such as enrofloxacin and florfenicol. This study provides a reference for exploring the impact of aquaculture in the Poyang Lake area on public health.

Tem Gene Detection as ESBL Marker in Clinical Isolates Klebsiella Pneumoniae

Klebsiella pneumoniae is one of the most common ESBL-producing microorganisms. The research aims to detect the Tem gene in clinical isolates of K. pneumonaie from the Laboratory of clinical pathology of Wahidin Sudirohusodo General Hospital. Methods: This research is a descriptive cross-sectional design. Fifty samples of K. pneumonieae were obtained from the Laboratory of clinical pathology from March to May 2018. Tem gene was detected using primary Tem 164.SE and Tem 165.AS (445 bp) of Polymerase Chain Reaction (PCR). The research results indicate that 48 isolates are positive, containing the Tem gene with the highest number found in the sputum specimen. Genotype detection of bacteria-producing ESBL is essential so that antibiotic therapy given to the patients is more effective and efficient.

Septin9 DNA methylation as a promising biomarker for cervical cancer

Aberrant Septin9 methylation in cervical cancer has been rarely studied. We aimed to identify its diagnostic value in cervical cancer using cervical scrapings, and its predictive potential in plasma for pelvic nodal metastasis of cervical cancer. The statuses of methylated Septin9 in fresh cervical lesions and cervical scrapings were first evaluated by using quantitative methylation-specific PCR. Subsequently, the relationship between Septin9 methylation in 113 plasma samples and pelvic nodal metastasis of cervical cancer was evaluated. Methylated Septin9 was detected in all cancerous tissues, but not in cervicitis. The degrees of Septin9 methylation increased with growing severity of cervical lesions in cervical scrapings. The sensitivity of methylated Septin9 was lower than that of cytology, while it yielded a high specificity and area under the curve in detecting high-grade squamous intraepithelial lesion or cervical cancer; and when Septin9 methylation combined with HPV16/18 genotyping, the sensitivity would increase from 70.42% to 82.39%. Plasma-based Septin9 methylation had a high discriminatory power in predicting pelvic nodal metastasis of cervical cancer, with an optimal specificity of 81.48%. In conclusion, we demonstrated methylated Septin9 to be an innovative diagnostic biomarker for cervical cancer and its non-invasive predictive potential in plasma for pelvic nodal metastasis of cervical cancer. Impact statement What is already known on this subject? The occurrence of cervical cancer is related to Septin9 methylation. In fresh specimens and cervical scrapings, we found the degrees of methylated Septin9 increased with growing severity of cervical lesions. Compared with HPV16/18 genotyping and cytological detection, Septin9 methylation had a better specificity and AUC in detecting ≥ HSIL. Furthermore, plasma-based Septin9 methylation also had a high specificity for pelvic lymphatic metastasis prediction. What the results of this study add? Methylation analysis of Septin9 indicated a similar sensitivity, specificity and AUC in detecting ≥ HSIL, relative to HPV16/18 genotyping. Compared with cytological method, Septin9 methylation also yielded a higher specificity and AUC in detecting ≥ HSIL. And we also found plasma-based Septin9 methylation had a high discriminatory power in predicting pelvic nodal metastasis of cervical cancer, with an optimal specificity of 81.48%; additionally an increasing sensitivity from 50% to nearly 80% was found when combined with SCCAg. What the implications are of these findings for clinical practice and/or further research? This study aimed to evaluate the relationship between Septin9 methylation and cervical cancer, and to explore the value of methylated Septin9 in the detection of cervical (pre)cancerous lesions. Moreover, we would explore plasma-based ctDNA biomarkers for pelvic lymphatic metastasis prediction of cervical cancer, to improve non-invasive predictive accuracy of pelvic nodal metastasis and reduce the complications caused by pelvic lymphadenectomy.

Distribution of emm, superantigen and other virulence genotypes and detection of phylogenetic relationships in group A streptococcal isolates.

Group A streptococci are important pathogens with various virulence factors, such as M protein, superantigens, hemolysins, deoxyribonuclease, and proteases. The aims of this study are to investigate the detection of emm genotypes and other virulence genes, such as SAgs, DNase, protease, antibiotic resistance, and phylogenetic relationships in GAS strains isolated from clinical samples.Test strains were obtained from Çukurova University Balcalı Hospital and regional hospitals in Adana province. The M proteins were detected by sequence analysis of emm genes. SAgs and other virulence gene profiles were determined using the Multiplex-PCR method. The antibiotic susceptibility of the isolates was performed by the disc diffusion method and evaluated according to CLSI criteria. The PFGE method was used to determine the clonal relationship between the strains.The emm gene was positive in 86 isolates. The most common emm genotypes were emm28 (22%), emm1 (18.6%), emm12 (13.9%), and emm3 (11.6%). Also, the most common virulence genes were speG (58.1%), speC (56.9%), sdaB (53.4%), and mac (53.4%). The rates of resistance to erythromycin, clindamycin, levofloxacin, ciprofloxacin and telithromycin were 19.8%, 16.3%, 4.7%, 3.5%, and 3.5%, respectively.As a result, additional regional studies on the detection and prevalence of GAS virulence factors in Turkey are required. We believe that this study will provide valuable information for epidemiological studies on emm sequences, Sags, and other virulence factors of Streptococcus pyogenes in Turkey.

Molecular Study of Adenovirus Genotypes 40 and 41 in Children with Acute Gastroenteritis.

Adenovirus is a common virus associated with acute gastroenteritis in children. There are certain genotypes that are prevalent in these infections, such as genotypes 40 and 41. The aim of the present study was to investigate the incidence of adenovirus genotypes 40 and 41 in children with acute gastroenteritis by polymerase chain reaction (PCR) and also to determine the possibility of Adenovirus co-infections with Rotavirus. The study was a cross-sectional study that included 100 children with acute gastroenteritis. The children were subjected to full history taking and clinical examination. Stool samples from the patients were subjected to detection of adenovirus and rotavirus antigens by enzyme-linked immunosorbent assay (ELISA) and detection of adenovirus genotypes 40 and 41 by polymerase chain reaction (PCR). The most prevalent virus by the used methods was rotavirus antigen in the stool (35%). Adenovirus antigen detection was positive in 23% of the stool samples, with positive PCR for these samples in 22%. The ADv40 was detected in 13 samples, and ADv41 was detected in 9 samples. One positive sample by adenovirus antigen ELISA was negative by PCR for these genotypes. Mixed rotavirus and adenovirus by ELISA were detected in 7% of the children. In patients with positive adenovirus antigen by ELISA, the most common symptoms were vomiting (54.5%) and abdominal pain (45.5%). An insignificant difference between fever (P=0.94) and abdominal pain (P=0.63) was detected in children infected with adenovirus compared to patients infected with other organisms. The adenovirus was detected in 68.2% of children with acute gastroenteritis ≤ 24 months. Vomiting was significantly increased in children with adenovirus (54.5%) compared to children negative for adenovirus (23.1%-P=0.004, OR 4.0, 95%CI: 1.5-10.8). The study highlights the presence of adenovirus genotypes 40 and 41 in the stool of children with acute gastroenteritis. Combined rotavirus and adenovirus infections were detected in our study.

An AS-qPCR-based method for the detection of Alzheimer's disease-related SNPs.

Alzheimer's disease (AD) is one of the most serious neurodegenerative diseases in the world and has a strong genetic predisposition. At present, there is still no effective method for the early diagnosis and prevention of AD. Accumulating evidence shows the association of several loci with AD risk, such as apolipoprotein E (APOE) and translocase of outer mitochondrial membrane 40 (TOMM40). However, for routine disease diagnosis in clinics, genotype detection methods based on gene sequencing technology are time-consuming and excessively costly. Thus, in this study, we developed a high-sensitivity, low-cost, and convenient single nucleotide polymorphism(SNP) detection assay method based on allele-specific quantitative polymerase chain reaction (AS-qPCR) technology, which can be used to determine the SNP genotype in APOE and TOMM40. A total of 40 patients were recruited from the outpatient department of the memory clinic of Dongzhimen Hospital, Beijing University of Chinese Medicine. The SNP detection assay method includes three steps. First, positive plasmids with different genotypes (TT/CC/TC) in APOE rs429358, rs7412, and TOMM40 rs11556505 were prepared. Second, 3'-T/3'-C primers were designed to amplify these positive plasmids for each SNP site. Finally, we calculated the log10 of the copy number ratio for each positive plasmid, and the genotype interpretation interval was established. Based on this method, we investigated whether the SNPs in 40 patients could be accurately calculated using AS-qPCR technology. The accuracy of SNP detection was verified by PCR-Pooling sequencing. The results showed that SNP genotypes assessed by AS-qPCR technology corresponded perfectly to the results obtained by conventional DNA sequencing. We have developed a genotype detection method for AD based on AS-qPCR, which can be performed easily, rapidly, accurately, and at low cost. The method will contribute to the early diagnosis of patients with late-onset Alzheimer's and the detection of large clinical samples in the future.

Association of HOXC8 Genetic Polymorphisms with Multi-Vertebral Number and Carcass Weight in Dezhou Donkey.

An increase in the number of vertebrae can significantly affect the meat production performance of livestock, thus increasing carcass weight, which is of great importance for livestock production. The homeobox gene C8 (HOXC8) has been identified as an essential candidate gene for regulating vertebral development. However, it has not been researched on the Dezhou donkey. This study aimed to verify the Dezhou donkey HOXC8 gene's polymorphisms and assess their effects on multiple vertebral numbers and carcass weight. In this study, the entire HOXC8 gene of the Dezhou donkey was sequenced, SNPs at the whole gene level were identified, and typing was accomplished utilizing a targeted sequencing genotype detection technique (GBTS). Then, a general linear model was used to perform an association study of HOXC8 gene polymorphism loci, multiple vertebral numbers, and carcass weight for screening candidate markers that can be used for molecular breeding of Dezhou donkeys. These findings revealed that HOXC8 included 12 SNPs, all unique mutant loci. The HOXC8 g.15179224C>T was significantly negatively associated with carcass weight (CW) and lumbar vertebrae length (LL) (p < 0.05). The g.15179674G>A locus was shown to be significantly positively associated with the number of lumbar vertebrae (LN) (p < 0.05). The phylogenetic tree constructed for the Dezhou donkey HOXC8 gene and seven other species revealed that the HOXC8 gene was highly conserved during animal evolution but differed markedly among distantly related animals. The results suggest that HOXC8 is a vital gene affecting multiple vertebral numbers and carcass weight in Dezhou donkeys, and the two loci g.15179224C>T and g.15179674G>A may be potential genetic markers for screening and breeding of new strains of high-quality and high-yielding Dezhou donkeys.

Revealing Genetic Diversity and Population Structure of Endangered Altay White-Headed Cattle Population Using 100 k SNP Markers.

Understanding the genetic basis of native cattle populations that have adapted to the local environment is of great significance for formulating appropriate strategies and programs for genetic improvement and protection. Therefore, it is necessary to understand the genetic diversity and population structure of Altay white-headed cattle so as to meet the current production needs under various environments, carry out continuous genetic improvement, and promote rapid adaptation to changing environments and breeding objectives. A total of 46 individual samples of endangered Xinjiang Altay white-headed cattle were collected in this study, including nine bulls and 37 cows. To collect genotype data, 100 k SNP markers were used, and then studies of genetic diversity, genetic structure, inbreeding degree, and family analysis were carried out. A total of 101,220 SNP loci were detected, and the genotype detection rate for individuals was ≥90%. There were 85,993 SNP loci that passed quality control, of which 93.5% were polymorphic. The average effective allele number was 0.036, the Polymorphism Information Content was 0.304 and the minimum allele frequency was 0.309, the average observed heterozygosity was 0.413, and the average expected heterozygosity was 0.403. The average genetic distance of Idengtical By State (IBS) was 0.3090, there were 461 ROH (genome-length homozygous fragments), 76.1% of which were between 1 and 5 MB in length, and the average inbreeding coefficient was 0.016. The 46 Altay white-headed cattle were divided into their families, and the individual numbers of each family were obviously different. To sum up, the Altay white-headed cattle conservation population had low heterozygosity, a high inbreeding degree, few families, and large differences in the number of individuals in each family, which can easily cause a loss of genetic diversity. In the follow-up seed conservation process, seed selection and matching should be carried out according to the divided families to ensure the long-term protection of Altay white-headed cattle genetic resources.

Identifying Predictors of Anal HPV Status in HPV-Vaccinated MSM: A Machine Learning Approach

ABSTRACT Anal human papillomavirus (HPV) infection has a high prevalence in men who have sex with men (MSM), resulting in an increased risk for anal cancer. The present work aimed to identify factors associated with HPV in a prospective cohort of HPV-vaccinated MSM using a random forest (RF) approach. This observational study enrolled MSM patients admitted to an Italian (sexually transmitted infection) STI-AIDS Unit. For each patient, rectal swabs for 28 different HPV genotype detection were collected. Two RF algorithms were applied to evaluate predictors that were most associated with HPV. The cohort included 135 MSM, 49% of whom were HIV-positive with a median age of 39 years. In model 1 (baseline information), age, age sexual debut, HIV, number of lifetime sex partners, STIs, were most associated with the HPV. In model 2 (follow-up information), age, age sexual debut, HIV, STI class, and follow-up. The RF algorithm exhibited good performances with 61% and 83% accuracy for models 1 and 2, respectively. Traditional risk factors for anal HPV infection, such as drug use, receptive anal intercourse, and multiple sexual partner, were found to have low importance in predicting HPV status. The present results suggest the need to focus on HPV prevention campaigns.

Development of field-applicable endogenous internally controlled recombinase-aided amplification (EIC-RAA) assays for the detection of human papillomavirus genotypes 6 and 11 using sample releasing agent

ObjectiveHuman papillomavirus (HPV) 6 and 11 are the two most common low-risk HPV subtypes, accounting for more than 90% of condyloma acuminatum. A simple, accurate and rapid screening method to be applied in community-level hospitals is in high demand. MethodsEndogenous internally controlled recombinase-assisted amplification (EIC-RAA) assays for HPV6 and 11 were performed in a single closed-tube at 39 °C within 30 min. The sensitivity and specificity of EIC-RAA were examined using recombinant plasmids and pre-tested HPV DNA. A total of 233 clinical samples were collected, and the DNA was extracted by traditional multi-step extraction, or sample releasing agent, before analysis by EIC-RAA. For comparison, HPV detection via Quantitative real-time PCR (qPCR) was also performed. ResultsThe sensitivity of EIC-RAA analysis was 10 copies/reaction for HPV6, 100 copies/reaction for HPV11, and 100 copies/reaction for the human β-globin gene. No cross-reaction was observed with other HPV subtypes. Clinical performance of the EIC-RAA assay achieved a 100% of concordance rate with the commercial HPV qPCR kit. Further, the EIC-RAA assay achieved a 100% of concordance rate when using multi-step extracted DNA and sample releasing agent-processed DNA. SummaryThe EIC-RAA assay for HPV6 and 11 detection possesses the advantages of accuracy, simplicity and rapidity, and demonstrates great potential to be used in community-level hospitals for field investigation.

Development of an HPV Genotype Detection Platform Based on Aggregation-Induced Emission (AIE) and Flow-Through Hybridization Technologies.

Genetic mutations can cause life-threatening diseases such as cancers and sickle cell anemia. Gene detection is thus of importance for disease-risk prediction or early diagnosis and treatment. Apart from genetic defects, gene detection techniques can also be applied to gene-related diseases with high risk to human health such as human papillomavirus (HPV) infection. HPV infection has been strongly linked to cervical cancer. To achieve a high-throughput HPV gene detection platform, the flow-through hybridization system appears to be one of the commercialized diagnostic techniques for this purpose. The flow-through hybridization technique is based on a vacuum-guided flow of DNA fragments which is continuously directed toward the oligoprobes that are immobilized on the testing membrane. However, the conventional colorimetric method and signal read-out approach suffers a problem of low sensitivity. On the contrary, fluorescence approaches allow more sensitive detection and broad sensing ranges. In this work, a fluorescent dye HCAP, which possesses aggregation-induced emission (AIE) properties and is responsive to alkaline phosphatase, was developed and applied to the flow-through hybridization platform to achieve HPV genome diagnosis of clinical samples. Also, an automatic membrane reader was constructed based on the AIE-based diagnosis platform which can identify the diagnostic result of patient DNA with a total concordance rate of 100% in the clinical trial.

Prevalence and Molecular Evaluation of Hepatitis C Virus Infection among Multi-transfused Thalassemia Patients in South of Iran.

ObjectivesInformation regarding the magnitude of hepatitis C virus (HCV) infection among thalassemia patients is of great importance for health care providers to assess blood safety and improve the quality of screening systems. Therefore, this study evaluated the prevalence, risk factors, and genotypic pattern of HCV infection among β-thalassemia patients in South Iran.MethodsThis descriptive-analytical cross-sectional study was conducted from March to June 2019. All patients with β-thalassemia major from Borazjan, Bushehr, Delvar, Kangan, and Ahram cities participated in the study and attended the transfusion center of the Bushehr University of Medical Sciences located in southern Iran. Serum samples were tested for the presence of anti-HCV antibodies by an enzyme-linked immunosorbent assay. The seropositive serum samples were tested for detection of HCV viremia and genotypes by semi-nested reverse transcriptase-polymerase chain reaction and sequencing.ResultsOf 125 thalassemia patients, 22 (17.6%) were positive for anti-HCV antibodies and two (1.6%) had HCV viremia with genotype 3a. HCV seroprevalence increased with age, so anti-HCV seropositive thalassemia patients had significantly higher mean age than anti-HCV seronegative patients. HCV seroprevalence was higher among female patients, residents of Kangan, patients with blood transfusion every two weeks, Fars patients, and thalassemia patients with alanine aminotransferase levels of < 20 IU/L and aspartate aminotransferase levels of > 80 IU/L. Nevertheless, anti-HCV seroprevalence among thalassemia patients was not statistically associated with these variables.ConclusionsThese results indicate ongoing HCV incidence among the thalassemia population in this region. Transfusion of HCV-seronegative viremic blood units donated during the infectious window period contributes to HCV infection in thalassemia patients. These findings highlight the need to include sensitive molecular assays in the screening process of donated blood for HCV infection in Iran.

A duplex fluorescent quantitative PCR assay to distinguish the genotype I and II strains of African swine fever virus in Chinese epidemic strains.

African swine fever (ASF) is a highly contagious hemorrhagic disease that affects domestic and wild pigs. A recent study reported that both ASF virus (ASFV) genotypes I and II have invaded farm-raised pigs in China, causing chronic infection and morbidity. To develop a duplex fluorescent quantitative PCR method to distinguish the ASFV genotypes I and II in Chinese epidemic strains, the probes and primers were designed based on the B646L sequences of genotypes I and II listed in the GenBank database. After optimizing the system, a duplex fluorescent quantitative PCR method for simultaneous detection of ASFV genotypes I and II B646L genes was successfully established. This method had no cross-reaction with Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), or Porcine Parvovirus (PPV), indicating that it has strong specificity. The sensitivity results indicated that the minimum detection limit of ASFV genotypes I and II B646L was 10 copies/Rxn. The inter- and intra-group coefficients of variation were both <3%, indicating that the method was highly reproducible. Therefore, the established duplex fluorescent quantitative PCR assay is important for the differential detection and epidemiological investigation of ASFV.

Multiple Stresses of Wheat in the Detection of Traits and Genotypes of High-Performance and Stability for a Complex Interplay of Environment and Genotypes

The effect of traits and the interaction of genotype × environment (GE) is one of the major challenges in detecting traits and genotypes with outstanding performance and stability through various stresses and years. The objective of this study was to identify the genetic influence traits of wheat, and genotypes with outstanding performance and stability under different environmental stress. The trials were carried out in two consecutive seasons with three treatments (optimal irrigation, limited irrigation, and heat stress), totaling six test environments at two different locations. After observing the importance of GE interaction, and the statistical significance for all studied traits, multivariate analysis was applied using stepwise regression (SR) for detecting influenced traits, and AMMI, AMMI’s stability values (ASV), yield stability index (YSI), superiority and GGE biplot methods to identify the genotype’s phenotypic stability. SR analysis showed that nine out of 22 traits have contributed significantly to grain yield (GY), which varied according to the environment. Equations of the models (GY) regression coefficient values reflected the importance seven of them have on a significant positive correlation on GY. The study confirmed the importance of AMMI and GGE biplots in decoding the GEI based on GY data. AMMI1 biplots showed that the three environments E1, E4, and E6 were the stronger interacting environments than E2, E3, and E5, in which the interaction was weak. YSI, superiority analysis, and superiority multi-trait analysis scores were largely compatible. YSI scores described the six genotypes viz, G5 (DHL26), G12 (DHL29), G10 (DHL01), G18 (Sakha-93), G2 (DHL02) and, G6 (Gemmeiza-9), these were marked by high stability and productivity. The GGE biplot analysis showed genotypes (G15 (Misr1) and G4 (DHL07)) recorded the highest grain yield in E3 and E4, whereas genotype G18 (Sakha-93) was in E6. It also showed G19 (Pavone-76) was the best genotype due to being situated in the center of the concentric circles and due to its high-yield. The methods considered were compatible with the detection of promising wheat genotypes with high mean performance and outstanding phenotypic stability across various stresses and years.

Phylogenetic analysis and genotyping of Iranian infectious haematopoietic necrosis virus (IHNV) of rainbow trout (Oncorhynchus mykiss) based on the glycoprotein gene.

Infectious haematopoietic necrosis (IHN) is known as one of the most contagious systemic viral diseases in salmonids which can lead to significant mortality rates and negative impacts on the salmonid farming industry. Infectious haematopoietic necrosis virus (IHNV) was first detected in rainbow trout (Oncorhynchus mykiss) farms in Iran in 2003. We conducted the present study to determine the detection of IHN genotypes in rainbow trout (O. mykiss) in farms in the central parts of Iran, using molecular and phylogenetic techniques. Samples were collected from fries exhibiting clinical signs such as darkening of the skin, abdominal swelling, and loss of appetite. Phylogenetic analysis was performed by the neighbour-joining method, using MEGA 5.1 software. For phylogenetic analysis and genotyping of IHNV from central parts of Iran, the sequences of the glycoprotein gene were determined for two Iranian isolates (Jahad-UT1 and Jahad-UT2). Phylogenetic analysis revealed that the detected strains (Jahad-UT1 and Jahad-UT2 isolates) are closely related (97.23%-100%) to European isolates within genogroup 'E'. This finding indicates that Jahad-UT1 and Jahad-UT2 isolates have been widely transferred to Iran from European countries. Moreover, the nucleotide diversity of these Iranian isolates showed a close relationship with the North American and Asian isolates, although the Iranian isolates were collected from a smaller geographical area and within a shorter time period between 2014 and 2015.

Development of a duplex real-time reverse transcription-polymerase chain reaction assay for the simultaneous detection of goose astrovirus genotypes 1 and 2

SummaryGoose astrovirus (GAstV) is a highly infectious pathogen that causes gout in goslings (<15 old) with typical symptoms of white urate disposition on the surface of the visceral organs and articular cavity, and a high mortality rate up to 50 %. To establish a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay for the rapid detection of the two GastV genotypes(GAstV-1 and GAstV-2), two pairs of primers and a pair of matching TaqMan probes were designed based on conserved regions of the ORF1b gene. The established duplex rRT-PCR assay showed no cross-reactivity with 10 other common waterfowl pathogens. The minimum detection limit was 10 copies/reaction for both GAstV-1 and GAstV-2. To validate the assay, 36 cloacal swabs from experimentally infected goslings and 33 field clinical samples were tested. The assay results of the experimentally infected goslings matched the infection scheme. The positive rates of GAstV-1 and GAstV-2 in the field clinical samples were 36.36 % and 54.55 %, respectively, and the co-infection rate of the two viruses was 21.21 % based on the duplex rRT-PCR assay. In conclusion, the established assay represents a specific, sensitive, and convenient tool for detecting GAstV-1, GAstV-2, and their co-infections, and for conducting epidemiological surveys.