Specific Polymerase Chain Reaction Research Articles

Investigation of meat species adulteration in beef-based meat products via real-time PCR in Türkiye

The addition of poultry and other meats to products made from 100% beef is prohibited in Türkiye. In addition, in Türkiye, where the majority of the population is Muslim, the use of pork and single-hoofed meat is a matter of concern due to religious reasons. This study utilized a sensitive and specific real-time polymerase chain reaction (PCR) method to identify the different meat species in meat products marketed as 100% beef and sold in the eastern Türkiye provinces of Erzurum, Erzincan, Kars, Ağrı, and Bingöl. The real-time PCR method was used to investigate the presence of DNA specific to six animal species (chicken, turkey, pork, horse, donkey, and camel). The analysis revealed no traces of horse, donkey, camel, or pork meat in any of the 100 samples of Turkish fermented sausage (sucuk), salami, and sausage. Contrary to the product labels claiming 100% beef content, poultry DNA residues were identified in a salami sample from Erzincan, while turkey DNA residues were found in sucuk samples from both Ağrı and Kars. The study’s findings reveal that contrary to label information, various types of meat can be present in meat products sold by both trademarked and local butchers. Consequently, this paper emphasizes the need for routine checks on market-sold products to ensure they comply with legislation and avoid consumer deception. Furthermore, the Real-Time PCR method, with its rapid results and high sensitivity, is deemed beneficial for food safety, consumer rights, and the prevention of unfair competition.

How to distinguish mpox from its mimickers: An observational retrospective cohort study.

During the current global outbreak of mpox (formerly monkeypox), atypical features were frequently described outside endemic areas, raising concerns around differential diagnosis. In this study, we included 372 adult patients who had clinical signs consistent with mpox and who were screened using non-variola orthopoxvirus specific quantitative polymerase chain reaction (PCR) between 15 May and 15 November 2022 at the University Hospital Institute Méditerranée Infection, Marseille, France. At least one clinical sample was positive for 143 (38.4%) of these patients and 229 (61.6%) were negative. Clinically, patients who had mpox presented more frequently with systemic signs (69.9% vs. 31.0%, p < 10-6 ) including fever (51.0% vs. 30.1%, p < 10-3 ), myalgia (33.5% vs. 17.9%, p = 0.002), and lymphadenopathy (38.5% vs. 13.1%, p < 10-6 ). Among the patients who were negative for the non-variola orthopoxvirus, an alternative diagnosis was identified in 58 of them (25.3%), including chickenpox (n = 30, 13.1%), syphilis (n = 9, 4%), bacterial skin infection (n = 8, 3.5%), gonococcus (n = 5, 2.2%), HSV infection (n = 5, 2.2%), and histoplasmosis (n = 1, 0.4%). Overall, in the current outbreak, we show that mpox has a poorly specific clinical presentation. This reinforces the importance of microbiological confirmation. In symptomatic patients who are negative for the monkeypox virus by PCR, a broad differential diagnosis should be maintained.

The role of methylenetetrahydrofolate reductase gene polymorphisms hypercoagulable status of coronavirus disease

Abstract Background: Hypercoagulation is a hallmark in coronavirus disease (COVID-19). The activity of the enzyme methylenetetrahydrofolate reductase (MTHFR) determines homocysteine levels, and polymorphisms in the enzyme’s gene can influence the enzyme activity with a consequence of hypercoagulability in patients with COVID-19. Objectives: To investigate the association of two single nucleotide polymorphisms (SNPs) in the MTHFR gene with hypercoagulability status in COVID-19. Materials and Methods: This is a retrospective cross-sectional study, which included 90 patients diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with variable severity. Patients were classified according to D-dimer level at admission into two groups: with and without hypercoagulability. Nucleic DNA was extracted from leukocytes and gene fragments corresponding to C677T and A1298C MTHFR gene were amplified and genotyped using allele specific polymerase chain reaction. Results: Hypercoagulation was reported in 42.22% of the patients. The mutant homozygous genotype (TT) was more frequent among hyper - than normocoagulable patients (13.6% vs. 1.92%) with a significant difference (odds ratio [OR] = 9.28, 95% confidence interval [CI] = 1.02–84.78, P = 0.048). Furthermore, T allele was more common among hyper- than normocoagulable patients (28.95% vs. 13.46%) with a significant difference (OR = 2.62, 95% CI = 1.24–5.5, P = 0.012). In contrast, the SNP A1298C had no significant impact. Conclusions: The TT genotype and T allele of C677T polymorphism but not A1298C in cMTHFE gene could be considered a risk factor for the hypercoagulable status in COVID-19.

Borrelia persica infection in wild carnivores in Israel: molecular characterization and new potential reservoirs

BackgroundBorrelia persica causes tick-borne relapsing fever in Israel, the eastern Mediterranean basin, and Asia. Relapsing fever is associated with severe illness and potentially death in humans and animals. Since B. persica infection has rarely been described in wild animals, the aim of this study was to evaluate the prevalence of infection with B. persica in wild carnivores in Israel.MethodsSpleen and blood clot samples from wild carnivores, which underwent necropsy, were tested for the presence of Borrelia DNA by real-time polymerase chain reaction (PCR). PCR products were sequenced, and the spirochete loads were quantified using a specific quantitative PCR (qPCR).ResultsA total of 140 samples from 74 wild carnivores were analyzed for the presence of Borrelia DNA. Six out of the 74 (8.1%) animals were found positive for B. persica by PCR and sequencing of the flagellin B gene, of which 4/74 (5.4%) were also positive by PCR for the glycerophosphodiester phosphodiesterase (glpQ) gene. Positive samples were obtained from three European badgers, and one striped hyena, golden jackal, and red fox each. All B. persica-positive animals were young males (P < 0.0001). Quantifiable results were obtained from 3/5 spleen and 4/5 blood samples. The spirochete loads in the blood were significantly higher than those found in the spleen (P = 0.034).ConclusionsThe prevalence of B. persica infection found in wild carnivores brought for necropsy was unexpectedly high, suggesting that this infection is widespread in some wild animal species in Israel. This is the first report of B. persica infection in the European badger and striped hyena. These carnivores have a wide geographical range of activity, and the results of this survey raise the possibility that they may serve as reservoir hosts for B. persica.Graphical

THE FIRST SURVEY OF BOVINE BABESIA SPECIES INFECTING YAKS (BOS GRUNNIENS) IN MONGOLIA.

Yak (Bos grunniens) farming is an important part of Mongolia's livestock industry. Yaks survive in harsh mountain environments; provide meat, milk, and wool; and serve as a mode of transportation. In Mongolia, yaks are frequently raised alongside other livestock animals such as cattle, Bactrian camels, sheep, goats, and horses. Recently, we demonstrated that Babesia bovis, Babesia bigemina, and Babesia naoakii-parasites with the potential to cause clinical bovine babesiosis-infect not only cattle but also Bactrian camels in Mongolia. However, yaks have never been surveyed for Babesia infections in this country. In the present study, we surveyed yaks in 8 Mongolian provinces: Bayankhongor, Bayan-Ulgii, Khovd, Khovsgol, Omnogovi, Ovorkhangai, Uvs, and Zavkhan. Blood samples were taken and deoxyribonucleic acid (DNA) was extracted from 375 yaks. Furthermore, Giemsa-stained thin smears were prepared from 315 of the 375 blood samples and then examined for the microscopic detection of Babesia parasites. Microscopy revealed that 34 (10.8%) of 315 blood smears were positive for Babesia parasites. All 375 DNA samples were then tested for B. bovis, B. bigemina, and B. naoakii infection using specific polymerase chain reaction assays. We observed that 238 (63.5%) yaks in all surveyed provinces and 8 (2.1%) yaks in 3 provinces (Bayankhongor, Bayan-Ulgii, and Omnogovi) were positive for B. bovis and B. bigemina, respectively. However, all yaks tested were negative for B. naoakii. This epidemiological survey, the first to report Babesia infection in Mongolian yaks, suggests that disease management strategies for yaks in this country should further address bovine babesiosis.

Bacterial Diversity and Antibiotic Resistance Genes Associated with the Different Farming Systems of Black Tiger Shrimp (Penaeus monodon) in Bangladesh

Microbial community inhabiting the intestine of the shrimp (Penaeus monodon) and their surrounding environments (e.g., water and sediment) is considered as a key contributing factor for the sustainable farming of shrimp. Indiscriminate application of antibiotics in aquaculture is a growing concern due to the emergence of antibiotic resistant bacteria (ARB), more specifically the antibiotic resistance genes (ARGs). The present study investigates the microbiome composition and 19 ARGs from four different shrimp farming systems; (i) cluster, (ii) extensive, (iii) semi-intensive, and (iv) improved extensive in the southwest coastal region of Bangladesh. In doing so, the study applied advanced 16S rRNA-based metagenomic sequencing to study the bacterial composition. Moreover, gene specific polymerase chain reaction (PCR) was employed to detect the ARGs in shrimp, water, and sediments of different farming systems. In the current study, bacteria from the phylum Proteobacteria and Firmicutes were predominant among the samples (n = 12) collected from the different farming systems followed by Actinobacteria, Bacteroidetes, and Cyanobacteria. Firmicutes was the predominant phylum in the gut of shrimp cultured in the cluster (relative abundance 53.33%) and semi-intensive (relative abundance 59.2%) culture systems. Results indicated that the bacterial community structure was significantly ( p &lt; 0.05 ) distinct among gut, sediment, and water samples as well as the farming systems. The shared operational taxonomic unit (OTU) in the sediment sample (16,495) was nearly double than the gut (7,931) and water (8,513) bacterial communities. The improved extensive farming system showed 1,289 (11.05%) shared OTUs among gut, sediment, and water followed by semi-intensive (6.87%), cluster (6.27%), and extensive (5.46%) farming system. Among the tested ARGs, sul1, cat, gyrA(C), tetA, tetC, tetX, ere(A), vanR, and dfrA1 were predominant in water and sediment samples. Semi-intensive farming system had the highest prevalence of ARGs (21.05%) while the lowest prevalence was found in extensive (5.26%) farming system. Overall, the study provides a comprehensive scenario of bacterial composition and growing emergence of ARGs in shrimp farming of Bangladesh. Therefore, the production strategy must focus on the alternatives of antibiotic for shaping the shrimp cultivation technique more sustainable.

Molecular Identification and Detection of Streptococcus pneumoniae Serotypes Isolated from the Cerebrospinal Fluid of Children with Suspected Meningitis: First Report From Alborz, Iran

Background: Pneumococcus causes various infections, some of which are life-threatening, including pneumonia, meningitis, otitis media, bacteremia, and sinusitis. Currently, more than 95 serotypes have been identified. Objectives: The purpose of this study was to isolate and determine Streptococcus pneumoniae serotypes from the cerebrospinal fluid (CSF) of children with suspected meningitis using the multiplex polymerase chain reaction (PCR) method. Materials and Methods: A total of 864 CSF samples were taken from children with suspected meningitis who were admitted to Imam Ali Hospital of Karaj for one year (January 2019 to January 2020). To collect positive S. pneumonia samples, the lytA gene was traced using specific primers and PCR techniques. Results: The results of this study indicated that only 16 patients have a pneumococcal infection (1.85%), of whom 50% have encapsulated pneumococci. Furthermore, serotypes 38 (12.5%), 7F (12.5%), 23F (25%), and 6A/B (50%) were the most common serotypes causing invasive pneumococcal infections in the included samples. Conclusion: Among the techniques for serotyping S. pneumoniae, the multiplex PCR technique is known as a fast, easy, and low-cost method that can serotype a large number of samples. The results also showed that the 13-valent pneumococcal conjugate vaccine can cover at least 50% of the strains that cause invasive pneumococcal diseases (IPDs), which are life-threatening, especially in children. Therefore, it is suggested that the healthcare administrators of Iran design and implement a public vaccination program.

Identification and Molecular Characterization of Shamonda Virus in an Aborted Goat Fetus in South Africa.

Viruses in the Orthobunyavirus genus, Peribunyaviridae family, are associated with encephalitis, birth defects and fatalities in animals, and some are zoonotic. Molecular diagnostic investigations of animals with neurological signs previously identified Shuni virus (SHUV) as the most significant orthobunyavirus in South Africa (SA). To determine if other orthobunyaviruses occur in SA, we screened clinical specimens from animals with neurological signs, abortions, and acute deaths from across SA in 2021 using a small (S) segment Simbu serogroup specific TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR). Positive cases were subjected to Sanger sequencing and phylogenetic analysis to identify specific viruses involved, followed by next-generation sequencing (NGS) and additional PCR assays targeting the medium (M) segment and the large (L) segment. In total, 3/172 (1.7%) animals were PCR positive for Simbu serogroup viruses, including two horses with neurological signs and one aborted goat fetus in 2021. Phylogenetic analyses confirmed that the two horses were infected with SHUV strains with nucleotide pairwise (p-) distances of 98.1% and 97.6% to previously identified strains, while the aborted goat fetus was infected with a virus closely related to Shamonda virus (SHAV) with nucleotide p-distances between 94.7% and 91.8%. Virus isolation was unsuccessful, likely due to low levels of infectious particles. However, phylogenetic analyses of a larger fragment of the S segment obtained through NGS and partial sequences of the M and L segments obtained through RT-PCR and Sanger sequencing confirmed that the virus is likely SHAV with nucleotide p-distances between 96.6% and 97.8%. This is the first detection of SHAV in an aborted animal in SA and suggests that SHAV should be considered in differential diagnosis for abortion in animals in Southern Africa.

Genetic Characterization of a Cohort of Italian Patients with Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency.

Defects in the steroid 21-hydroxylase gene (CYP21A2) cause 21-hydroxylase deficiency (21OHD), the main cause of congenital adrenal hyperplasia (CAH). The disease shows a broad spectrum of clinical forms, ranging from severe or classical (salt wasting, SW, and simple virilizing, SV), to mild late onset or nonclassical (NC). 21OHD affects 1 in 15,000 in its severe classic form and 1 in 200-1000 in its mild NC form. There are many studies reporting the frequency of CYP21A2 pathogenic variants in different populations; however, few of them provide comprehensive information about Italian patients. Here, we present genetic results from a cohort of 245 unrelated Italian individuals with clinical diagnosis of CAH due to 21OHD. A specific polymerase chain reaction (PCR) protocol combined with Sanger sequencing was used for CYP21A2 analysis. The multiplex ligation-dependent probe amplification (MLPA) assay was employed for copy number variation (CNV) determination. One hundred fourteen (46.5%) of the index cases had the NC form, 57 (23.3%) had the SV form, and 74 (30.2%) presented the SW form of the disease. The most prevalent variant found in NC patients was the p.Val282Leu (51.3%), while the most frequent variants in the classical form were p.Ile173Asn (8.6%) and c.293-13C>G (26.0%). In our study, the frequency of large rearrangements was 15.3%, with CAH-X alleles representing 40% of all DEL/CONV. In addition, 12 alleles carried rare variants, and 1 had a novel variant p.(Arg342Gln). We observed phenotype-genotype correlation in 94.7% of cases. A complete concordance was observed in Groups 0 (enzyme activity completely impaired) where all patients had the SW form as expected. In Group A (0-1% residual enzyme activity), 78.4% of patients had the anticipated SW form while 21.6% were diagnosed with the SV form. Within Group B (~2% residual enzyme activity), 93.4% of patients exhibited SV form and 6.5% SW disease. Finally, 92.6% and 7.4% of patients belonging to Group C (enzyme partially impaired to ~20-60% residual activity) exhibited NC and SV phenotypes, respectively. This work, representing a comprehensive genetic study, expanded the CYP21A2 variants spectrum of Italian patients with 21OHD and could be helpful in prenatal diagnosis and genetic counseling.

Models for prediction of single base primer extension efficiency from position and type of single mismatch in primer-template duplex

Amplification and specificity of polymerase chain reaction (PCR) are affected by the position and type of primer-template mismatches (MMs) as well as various conditions of reaction. In this study, multiple linear regression (MLR) models and artificial neural network (ANN) models were developed for the prediction of the effects of primer-template mismatches on the primer extension efficiency in primer-template duplex. In MLR models, the independent variable Pi representing the position effect of i-th mismatch from 3′ end of primers was normalized to values between 0 and 1 according to the size of ΔΔGi, the difference of Gibbs free energy changes between the mismatch and its corresponding perfect-match, and other independent variables Pj representing the position effect of the j-th perfect-match from 3’ end of primer were coded 1. A dependent variable of MLR model was relative extension efficiencies of primers. In ANN models, an input layer has neurons equal to the number of independent variables of corresponding MLR models and a hidden layer and an output layer have four and one neurons, respectively. Our MLR models and ANN models outperform the previous polynomial regression model for the prediction of the single base extension (SBE) efficiencies of single-MM primers. Especially, ANN model 6 which has 32 neurons representing the position effect of mismatch, the type of mismatch and the annealing temperature on primer-template duplex in the input layer can predict the SBE efficiencies of single-MM primers with a high accuracy, since its correlation coefficients R in training set, testing set and all data are 0.9870, 0.9782 and 0.9857, respectively. These results will have a good prospect applicable to the design of primer and testing the primer specificity in genome database.

Effects of company and season on blood fluke (Cardicola spp.) infection in ranched Southern Bluefin Tuna: preliminary evidence infection has a negative effect on fish growth.

Aporocotylid blood flukes Cardicola forsteri and C. orientalis are an ongoing health concern for Southern Bluefin Tuna (SBT), Thunnus maccoyii, ranched in Australia. Therapeutic application of praziquantel (PZQ) has reduced SBT mortalities, however PZQ is not a residual treatment therefore reinfection can occur after the single treatment application. This study documents the epidemiology of Cardicola spp. infection in ranched SBT post treatment over three ranching seasons (2018, 2019 and 2021). Infection prevalence (percentage of SBT affected) and intensity (parasite load) was determined by adult fluke counts from heart, egg counts from gill filaments and the use of specific quantitative polymerase chain reaction (qPCR) for detection of C. forsteri and C. orientalis ITS-2 DNA in SBT hearts and gills. SBT Condition Index decreased as intensity of Cardicola spp. DNA in SBT gills increased, suggesting blood fluke infection had a negative effect on SBT growth (Spearman's r = -0.2426, d.f. = 138, p = 0.0041). Prevalence and intensity of infection indicated PZQ remained highly effective at controlling Cardicola spp. infection in ranched SBT, 10 years after PZQ administration began in this industry. Company A had the highest prevalence and intensity of Cardicola spp. infection in 2018, and Company G had the highest in 2019. No consistent pattern was seen in 2021. Overall, intensity of infection did not increase as ranching duration increased post treatment. Results from this study improve our knowledge of the biology of blood flukes and helps the SBT industry to modify or design new blood fluke management strategies to reduce health risks and improve performance of SBT.

Emergence of New Delhi metallo-β-lactamase-1 (NDM-1) producing Enterobacterales from water sources: An impending public health challenge in Adamawa-north senatorial zone, Nigeria

Background: The emergence of New Delhi metallo-beta-lactamase-1 (NDM-1) among Enterobacterales in water sources has raised a major public health concern and constitute critical threat to human health as these organisms exhibits high level of resistance to available potent antibiotics. The aim of this study is to detect the presence of NDM-1 gene among carbapenem resistant Enterobacterales (CRE) isolates from water sources.&#x0D; Methodology: A total of 256 water samples were collected from randomly selected hand-dug wells (128 samples) and river/stream (128 samples) for each of dry and rainy seasons in four out of the five local government areas (LGAs) of Adamawa-north senatorial zone, Nigeria. The water samples were filtered using membrane filtration technique and the filters introduced into appropriate bacteriologic media for bacterial growth. The bacterial isolates recovered were identified by both phenotypic and molecular protocols. Phenotypic carbapenem (imipenem) resistance was determined by disc diffusion test, blaNDM-1 gene was detected by specific polymerase chain reaction (PCR) test, and plasmid DNA was extracted and electrophoresed by standard procedure.&#x0D; Results: Of the 256 water samples analyzed for bacteria growth, 300 bacterial isolates of the order Enterobacterales were recovered. Of these, only 45 (12.6%) isolates were phenotypically resistant to carbapenem (imipenem) antibiotic and blaNDM-1 gene was detected in 30 (66.7%) of these. While blaNDM-1 gene was detected in all the isolates of Klebsiella oxytoca, Klebsiella variicola, Enterobacter aerogenes, Enterobacter hormaechei, Enterobacter asburiae, Citrobacter freundii, and Morganella morganii that were resistant to imipenem, other isolates harbored blaNDM-1 gene in varying proportion. Most of the isolates positive for blaNDM-1 also harbored R-plasmids.&#x0D; Conclusion: Emergence of carbapenem resistance mediated by NDM-1 gene in Enterobacterales isolated from water sources constitutes an emerging public health challenge with potential transmission to humans, thereby complicating the treatment of infections caused by these resistant pathogens in man. As such, the urgent need for antimicrobial surveillance and stewardship is of utmost importance.

Biosensor for PCR amplicons by combining split trehalase and DNA-binding proteins: A proof of concept study

The use of polymerase chain reaction (PCR) technology in low-cost settings has gained significant attention due to its ability to amplify and detect specific bacterial pathogen genes, aiding in the diagnosis of infectious diseases. PCR amplicons can be visualized by conventional endpoint agarose gel electrophoresis and fluorochrome-enabled real-time PCR. However, this is not practical in on-field tests due to cumbersome instrumentation, labor-intensive reaction preparation, and long time-to-results. Many studies have combined microfluidic devices or electrochemical dyes with PCR technology to enhance in-field operability. However, the high cost of manufacturing high-precision microfluidic chips and the dependence on non-portable readout equipment limit their further development. In this paper, we present a proof-of-principle study of a novel method combining split enzyme technology and DNA-binding proteins for the convenient and efficient detection of amplified genetic material from bacterial pathogens. The amplicon binding split trehalase assay (ABSTA) relies on incorporating specific recognition sequences of DNA-binding protein SpoIIID in tandem within one of the PCR primers. Applied by a Gram-type specific PCR assay, ABSTA was capable of discriminating Staphylococcus devriesei and Escherichia coli in <90 min after colony PCR amplicons bound split trehalase fragments-fused SpoIIID and triggered split enzyme complementation. The salt concentration, protein reagents versus DNA substrate ratio, direction and linker length of tandem recognition sites required for the complementation were optimized. The glucose produced by restored enzymatic activity was detectable by glucometer. With limited requirements for reaction preparation and the compatibility of ABSTA with commercially available handheld glucometers, this test platform has substantial potential to be implemented into a future point-of-care (POC) diagnostic tool for detecting pathogen specific genes with further improvement.

The association of PON1 and NOS3 genetic variants with the severity of COVID-19

The pandemic of coronavirus disease 2019 (COVID-19) is a global pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The aim of this study was to investigate the association of single nucleotide polymorphisms (SNPs) of paraoxonase 1(PON1) and nitric oxide synthase 3 (NOS3) genes with the severity of COVID-19. Study subjects were divided into mild and severe groups. Genotyping was conducted by allele specific real-time polymerase chain reaction (RT-PCR). Gene-gene interaction was assessed by multifactor dimensionality reduction (MDR) analysis. The results showed a significant association of both PON1 rs662 (A575G), and NOS3 rs2070744 (T786C) with the severity of COVID-19 (p = 0.0026, and p = 0.033, respectively). PON1 575AG (OR = 0.30, 95 % CI [0.13–0.68]) and NOS3 786TC (OR = 0.35, 95 % CI [0.15–0.78]) were associated with mild COVID-19 symptoms. The resulted SNP-SNP interaction model by MDR analysis was statistically significant (p = 0.0003). NOS3 786C * PON1 575G haplotype was significantly associated with mild cases of COVID-19 (p = 0.045).

Detection of resistance mutations in patients with anaplastic lymphoma kinase-rearranged lung cancer through liquid biopsy.

Tyrosine kinase inhibitors (TKIs) significantly improve clinical outcomes in patients with non-small cell lung cancer due to anaplastic lymphoma kinase (ALK) gene rearrangement. However, the rate of relapse with TKIs is high owing to the development of resistance mutations during treatment. Repeated biopsies during disease progression are crucial for elucidating the molecular mechanisms underlying the development of resistance to ALK inhibitors. Analysis of cell-free DNA (cfDNA) obtained from plasma is a novel approach for tumor genotyping. In this mixed prospective and retrospective observational cohort study, we investigated the clinical feasibility of continuous quantitative monitoring of ALK-acquired mutations in plasma obtained from patients with ALK+ non-small cell lung cancer by using a highly sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay. We enrolled nine patients, including three treatment-naïve patients recently diagnosed with ALK+ non-small cell lung cancer via tissue biopsy and expected to receive ALK TKIs and six patients already receiving ALK TKIs. Plasma samples were collected from these patients every 3 months. cfDNA was extracted from 66 samples during the study period, and 10 ALK mutations were simultaneously evaluated. The numbers of samples showing the G1202R, C1156Y, G1269A, F1174L, T1151ins, and I1171T mutations were 32, 16, 5, 4, 1, and 1, respectively. The L1196M, L1152R, V1180L, and S1206Y mutations were not detected. Correlation analyses between progression-free survival and the time from treatment initiation (or treatment modification) to the detection of resistance mutations revealed that although resistance mutations may occur before a drug change becomes necessary, there is a duration during which the disease does not progress. Our findings suggest that real-time quantitative monitoring of ALK resistance mutations during the response period could provide a time course of changes while acquiring resistance mutations. This information would be beneficial for designing an appropriate treatment strategy.

Distinguishing between Artemisia stolonifera and A. argyi by specific PCR of leaves and non-glandular trichomes

Artemisia stolonifera is a relative of A. argyi. The two species are difficult to be distinguished due to the similarity in leaf shape and have even less distinctive features after processing. This study aims to establish a method to quickly distinguish between them. At the same time, we examined the reasonability and applicability of the specific polymerase chain reaction(PCR) method. The C/T single nucleotide polymorphism was detected at the position 202 of the sequence, based on which specific primers were designed to identify these two species. The PCR with the specific primer JNC-F and the universal primer ITS3R produced a specific band at 218 bp for A. argyi and no band for A. stolonifera, which can be used to detect at least 3% of A. argyi samples mixed in A. stolonifera samples. The PCR with the specific primer KY-F and the universal primer ITS3R produced a specific band at 218 bp for A. stolonifera and no band for A. argyi, which can be used to detect at least 5% of A. stolonifera samples mixed with A. argyi. The limit of detection of the established method was 5 ng DNA. The established PCR method can accurately distinguish between A. stolonifera and A. argyi, which provides an experimental basis for the quality control of A. stolonifera and determines whether the herbs are adulterated.

The correlation between TLR9 (rs148805533 Del/Ins) gene polymorphisms and susceptibility to pulmonary tuberculosis

Abstract Background: One of the main infectious causes of morbidity and mortality worldwide is Mycobacterium tuberculosis. The Toll-like receptor (TLR) genes’ polymorphisms and mutations have been linked to an increased risk of infection in general. Objectives: This study aimed to investigate the association among TLR9 (rs148805533 Del/Ins), gene polymorphism, and pulmonary tuberculosis (PTB). Materials and Methods: Blood and sputum samples were collected from 70 patients with PTB and 30 healthy as a control group. Patients were diagnosed clinically by the specialized physician, in addition to use the acid-fast smear and culture on Lowenstein–Jensen. In addition, the molecular diagnostics technique was used by Gene Xpert device. The genotyping was carried out by using allele specific-polymerase chain reaction technique, and the TLR9 concentration was estimated by enzyme-linked immunosorbent assay (ELISA) technique. Results: The results of TLR9 polymorphism showed that the Ins/Ins allele, Del/Ins allele, and Del/Del allele genotypes frequencies at the site of (rs148805533) polymorphisms were significantly higher in PTB patients than the control groups (P = 0.05). The Ins/Ins allele genotype was the most frequent in PTB patients. In addition, the insertion allele was the most frequent in PTB patients and control groups. Conclusion: Our results indicated that in a sample of the local Iraqi population, the TLR9 (rs148805533) gene polymorphism may be a significant genetic determinant for PTB susceptibility.

Characteristics of Vitamin D3 Receptor Genotypes in T2DM of Iraqi Obese Women

Objective: This study aims to examine the association between the (rs1544410) polymorphism of the VDR gene with the pathogenesis of T2DM in Iraqi obese women.&#x0D; Methods: A case-control study was performed on 50 patients with T2DM and 50 apparently healthy subjects who were admitted to Al-Hussein Teaching Hospital and Al-Hassan Center of Diabetes and Endocrinology unit / Kerbala health directorate – Iraq during (April 2022 – March 2023). The T2DM groups were divided into two groups, 25 obese and 25 non-obese; the control group was divided into 25 obese and 25 non-obese as apparently healthy groups. The ELISA Kit was used to measure serum 25(OH)D3, heat shock protein-70, VDBP, insulin and C-peptide. Also HbA1c% and insulin resistance (HOMA-IR) were evaluated. The vitamin D3 receptor gene (VDR) variant and the SNP (rs1544410) polymorphism was determined using allele specific polymerase chain reaction, 1.5% agarose gel electrophoresis and then visualized by gel photo-documentation system.&#x0D; Results: The result of vitamin D3 variants genotype (rs 1544410) was a clear band with a molecular size of 200 bps. The size of the amplicon was determined by compare with DNA ladder 100 - 1500 bp. The result of the comparison between observed and anticipated values for SNIP with (rs 1544410) in the tested population was statistically significant, P= &lt; 0.001 and the difference between demographic characteristics and (rs 1544410) SNP, age and BMI shows non-significant difference among all groups. The difference between biomarkers and (rs1544410) SNP was performed using one-way ANOVA test to compare the mean levels of HSP-70, VDBP, C-peptide, RBC and HbA1c% which shown a non-significant difference among the variants of VDBP Genotype (rs1544410) in obese women (patients and control) studied groups, p value &gt; 0.05. &#x0D; Conclusion: The logistic analysis of the (rs1544410) SNP of the patients concluded that HSP-70, VDBP, and C-peptide level was no significantly related to the also C-peptide, was shown to be a related risk factor to both CT and CT alleles (1.003, p &gt; 0.05) in comparison with CC alleles. Furthermore, HbA1c% level was demonstrated to be related as a risk factor for the CG allele in comparison with CC and GG alleles (1.009, p &lt; 0.05).

Introgression of sharp eyespot resistance from Dasypyrum villosum chromosome 2VL into bread wheat

Wheat sharp eyespot, a stem disease caused by the soilborne fungus Rhizoctonia cerealis van der Hoeven, has become a threat to wheat production worldwide. Exploiting resistance resources from wild relatives of wheat is a promising strategy for controlling this disease. In this study, a new wheat–Dasypyrum villosum T2DS·2V#4L translocation line in the background of Chinese Spring (CS) showed stable resistance to R. cerealis. Introgression of the T2DS·2V#4L chromosome into wheat cultivar Aikang 58 by backcrossing produced a marked increase in sharp eyespot resistance in NIL-T2DS·2V#4L in comparison with NIL-T2DS·2DL, and no detrimental effects of 2V#4L on agronomic traits were observed in the BC2F2, BC2F2:3, and BC2F2:4 generations. Flow-sorted sequencing of 2V#4L yielded 384.3 Mb of assembled sequence, and 8836 genes were predicted of which 6154 had orthologs in at least one of the 2AL, 2BL, and 2DL arms of CS, whereas 1549 genes were unique to 2V#4L. About 100,000 SNPs were detected in genes of 2V#4L and 2DL in 10 sequenced bread wheat cultivars. A Kompetitive Allele Specific Polymerase chain reaction and 30 conserved ortholog sequence markers were developed to trace the 2V#4L chromatin in wheat backgrounds. T2DS·2V#4L compensating translocation lines represent novel germplasm with sharp eyespot resistance and the markers will allow rapid detection in breeding programs.

A specific and sensitive droplet digital polymerase chain reaction assay for the detection of tilapia lake virus in fish tissue and environmental samples.

Tilapia lake virus (TiLV) causes high mortality in farmed and wild tilapia in various countries. We developed a highly specific and sensitive droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify TiLV. The ddPCR assay could detect the virus at a lower threshold than the reverse transcription-quantitative polymerase reaction (RT-qPCR) method, and the sensitivity of the ddPCR assay was 10-fold higher. The diagnostic sensitivity and specificity of the ddPCR assay were 100% and did not cross-react with tilapia tissues infected with Tilapia parvovirus, Infectious spleen and kidney necrosis virus, Aeromonas hydrophila, Streptococcus agalactiae, S. iniae and Francisella noatunensis. The assay reproducibility was demonstrated by a high correlation coefficient of 0.998, and the inter-assay coefficients of variability indicated that the ddPCR assay exhibited low variability within and between measurements. The detection limit of the TiLV ddPCR assay was 100 fg cDNA, which is equal to 3.3 copies of TiLV. Furthermore, the ddPCR assay could detect TiLV in mucus, water and infected tissue samples and the lowest copy number of TiLV detected in water samples by the ddPCR assay was 7.9 ± 0.99 copies/reaction The results of the clinical samples tested for TiLV revealed that the ddPCR assay had a relatively higher detection rate than the RT-qPCR method. Overall, the ddPCR method offers a highly promising approach for the absolute quantification of TiLV in carrier fish and samples from the environment with low viral concentrations.