PCR Sequencing Methods Research Articles

Correlation of (IL-10) gene rs 1800896 polymorphism and tuberculosis risk in Iraq's Nineveh city

The equilibrium between host immune factors and bacterial evasion methods is critical for tuberculosis (TB) control. One immunomodulatory factor that has been suggested to affect a person's vulnerability to tuberculosis is interleukin-10 (IL-10).The study aimed to Identify: (1) Association of (IL-10) gene rs 1800896 polymorphism and the hazard of TB infection, (2) a novel variation of this gene that may be influencing its activity, (3) Association of some haematology tests and tuberculosis. The study included 334 patients with tuberculosis and control. DNA was extracted from blood samples, (IL-10) gene rs 180096 polymorphism was detected by Tetra ARMS PCR and gene sequencing method. (IL-10) levels were detected by Elisa test. Sequencing results for amplifying of the (IL-10) gene in tuberculosis patients revealed the existence of several nucleotide sequence variations that have not been reported before. Both a new phenotype of the (IL-10) protein and a new genotype for the (IL-10) gene were discovered in Nineveh, Iraq, at the National Center for Biotechnology Information. The ARMS PCR reaction results indicated a correlation between the genomic variation of the (IL-10) gene at the site (rs 180096) and tuberculosis patients. The genetic variation was observed in three different genotypes with varying proportions. The native allele C had a higher allelic frequency (65%) than the mutant allele T (35%). The study concluded that individuals with the (IL-10) gene rs180096 TT genotype under the dominant model were more likely to be at risk of TB infection.

First report of Ralstonia pseudosolanacearum phylotype I sequevar 14 causing bacterial wilt on tomato (Solanum lycopersicum) and eggplant (Solanum melongena) in North Carolina, USA.

Ralstonia solanacearum species complex (RSSC) consists of three species, including R. solanacearum, R. pseudosolanacearum, and R. syzygii. The K60-type strain of R. solanacearum was isolated from a wilted 'Marglobe' tomato in Raleigh, North Carolina (NC) in 1953 (Kelman 1954). It is classified as phylotype IIA, sequevar 7 (Prior and Fegan 2005). In July 2023, during a field visit in Eastern NC, patches of >50 eggplant (Solanum melongena cv. Pingtung Oriental) and tomato (Solanum lycopersicum cv. Saybrook) plants showing wilt symptoms were observed in a 1 ha field. Two plants from each host were collected from this site and tested positive for bacterial streaming in sterile deionized water for 2 min. One plant from each host was used for bacterial isolation by plating a 10 μl aliquot of the resulting bacterial streaming suspension on triphenyl tetrazolium chloride (TZC) medium (Kelman 1954) and incubated at 28°C for 48 hr. Multiple fluidal white colonies with a pink center and irregularly round morphology reminiscent of strains in the RSSC were observed on all plates. Only one colony from each plant host, NG-RL and EP-RL from tomato and eggplant, respectively, was selected for molecular characterization. Neither strain amplified the 357 bp band and was not R. solanacearum Select Agent (Opina et al. 1997). Genomic DNA from both NG-RL and EP-RL generated the 280 bp and 144 bp bands and confirmed as R. pseudosolanacearum phylotype I using the RSSC multiplex PCR (Fegan and Prior 2005). To determine sequevar, the primers Endo-F/Endo-R (Poussier et al. 2000; Fegan and Prior 2005) were used to sequence the partial endoglucanase (egl) gene from EP-RL and NG-RL (GenBank accessions: PQ554799 and PQ554800). These sequences were compared to publicly available egl sequences from GenBank and Cellier et al. (2023). A maximum likelihood phylogenetic tree showed that both NG-RL and EP-RL clustered with reference strains PSS81, MLI71-15, and Zo4 with 100% identity, confirming NG-RL and EP-RL are R. pseudosolanacearum phylotype I sequevar 14. To fulfill Koch's postulates, NG-RL and EP-RL inoculant was prepared from 48 h cultures grown on TZC plates at 28°C. Plates were flooded with sterile deionized water and then transferred to a falcon tube, adjusting O.D. 600 to 0.2 (~1×108 CFU/ml). The roots of six-week-old eggplants (cv. Black Beauty) and tomatoes (cv. Bonny Best) were wounded by running a scalpel through the soil 2 cm from the stem. This was repeated on six plants for both NG-RL and EP-RL. Mock-inoculated plants treated with sterile deionized water (SDW) served as controls. Plants incubated at 28°C in the greenhouse showed bacterial wilt symptoms nine days post-inoculation. R. pseudosolanacearum phylotype I sequevar 14 was confirmed from all bacteria-inoculated plants using the multiplex PCR and egl sequencing methods described above. No symptoms or bacteria were isolated from SDW mock-inoculated plants. There have been no prior reports of R. pseudosolanacearum phylotype I in North Carolina. To our knowledge, this is the first report on R. pseudosolanacearum phylotype I and the sequevar 14 in both tomato and eggplant in NC. This phylotype I is endemic to Asia and was first reported in the US in 2003 on pepper in Florida (Ji et al. 2006) and in 2015 on tomatoes in Louisiana (Jimenez Madrid et al. 2019). This finding highlights the need for a nationwide RSSC survey program, which can undoubtedly inform pathogen spread and management for crops in NC and beyond.

A TaqMan-MGB real-time PCR for discriminating between MS-H-live vaccine and field Mycoplasma synoviae strains.

Mycoplasma synoviae (MS) is an important pathogen in the poultry industry and has caused significant economic losses. Worldwide, the use of live attenuated vaccine for the MS-H strain has increased to prevent MS infection. However, there is no test available to discriminate the MS-H vaccine strains from the MS strains that are causing field infection. In this study, a TaqMan-MGB real-time PCR method (qPCR) was established, validated, and evaluated to discriminate between MS-H-live vaccine and field strains based on nucleotide differences in the hlyC gene. The validation was performed for sensitivity and reproducibility by constructing recombinant plasmids. The limits of detection were 1.07 × 101 copies/µL for the MS-H and 1.95 × 101 copies/µL for field strains, respectively. The intra- and inter-assay results were less than 2.5% based on the reproducibility test. No cross-amplification signals from other common chicken pathogens were detected. Thus, our data indicated that this qPCR is sensitive, specific, and reproducible. In addition, 709 chicken clinical samples were used to evaluate this qPCR test. The results showed that positive signals could be detected from the chicken choanal cleft swabs and are 100% in concordance with the PCR sequencing method. To the best of our knowledge, we found for the first time that both L- and C-type field MS were present in flocks immunized against the MS-H vaccine strain during the validation process. In addition, this is the first report of a field strain of C-type in China.IMPORTANCEMycoplasma synoviae (MS) is an important pathogen in the poultry industry and has caused significant economic losses. Worldwide, an increasing number of farms are using the live attenuated vaccine MS-H strain to prevent MS infections. In order to monitor vaccinated and naturally infected flocks and to continue the MS control and eradication program, a differentiation of infected from vaccinated animals (DIVA) test for MS is urgently needed. We developed a TaqMan-MGB real-time qPCR (qPCR) method with a pair of primers and two competitive TaqMan-MGB probes. We performed an evaluation that can discriminate between the MS-H-live vaccine and field MS strains based on nucleotide differences in the hlyC gene. It has great sensitivity and reproducibility, and greater specificity than other methods which were established by SNP sites of the obg gene and oppF gene.

Analysis of relative genes expression and mutation of pstB and efpA efflux pumps in Mycobacterium simiae isolates from suspected tuberculosis patients by using quantitative Real-time PCR

BackgroundMycobacterium simiae is commonly found in people with weak immune systems such as the elderly and people with tuberculosis and other lung diseases. The aim of this study is the epidemiology of M. simiae infections in Iran and the world, therefore, in this study, analysis of relative gene expression and mutation of pstB and efpA efflux pumps in Mycobacterium simiae isolates from suspected tuberculosis patients by using Real-time quantitative PCR. Sixty-five sputa samples of suspected tuberculosis specimens were collected. The identification of NTM Species by PCR sequencing and determining drug sensitivity by micro broth dilution method. The investigate mutations in pstB and efpA efflux pump genes using the PCR-sequencing method. Comparative evaluation of the replication efficiency of internal control gene 16SrDNA and target genes pstB and efpA.ResultsIn total, 15 isolates of M. simiae were evaluated and a drug sensitivity test was performed for them against the antibiotic ethambutol, moxifloxacin, clarithromycin, and linezolid. The highest resistance to moxifloxacin and linezolid antibiotics and the lowest resistance to clarithromycin antibiotics were observed. The results of expression levels of two efflux PstB and efpA showed that there was a significant difference in the expression level of the efpA efflux pump gene in M. simiae three-resistant and double-resistant isolates compared to the sensitive group and the standard.ConclusionsThe high level of antibiotic resistance In M. simiae isolates is an alarm and monitoring all factors related to antibiotic resistance, including efflux pumps, is an important research topic.

The distribution of pigeon adenoviruses in Northern Chinese pigeon and turtledove flocks provides further evidence of viral crosstransmission.

To understand the prevalence, genetic diversity, and potential pathogenicity of adenoviruses present in pigeon and turtledove populations. Nested PCR and Sanger sequencing methods were used to identify the genotype and percentage of various adenoviruses in the feces of pigeon (Columba) and turtledove (Streptopelia) populations. In Beijing, China, a total of 194 fresh feces samples from meat-use pigeons (C livia domestica), homing pigeons (C livia domestica), wild pigeons (C livia domestica), and turtledoves (S decaocto and S chinensis) were collected using noninvasive sampling collection techniques. Their partial DNA-dependent DNA polymerase gene sequences were obtained using nested PCR and double-ended Sanger sequencing, and their genotypes were then ascertained based on sequence alignment. A total of 6 genotypes of adenovirus were detected in pigeon and turtledove flocks, including pigeon adenovirus (PiAdV)-1, PiAdV-2A, PiAdV-3, PiAdV-4, PiAdV-5, and a novel adenovirus genotype (PiAdV-6). Among them, PiAdV-1 was found widespread in flocks of pigeons exhibiting extensive presentations of hepatic necrosis. Highly conserved PiAdV-4 and PiAdV-5 were found to be nonpathogenic and extensively distributed in all pigeon and turtledove groups. These findings imply the presence of diverse PiAdVs in pigeon and turtledove flocks, and the wild pigeons and wild turtledove birds are potentially serving as natural sources of these viruses. This study provides supportive evidence of the pathogenicity of different genotypes of adenovirus in pigeon flocks and also implies that stopping the transmission of the virus brought by wild pigeons and turtledoves may be important for the prevention of diseases associated with PiAdVs.

Development of a one-step multiplex RT-qPCR method for rapid detection of bovine diarrhea viruses.

Viral calf diarrhea poses a significant challenge to the cattle industry worldwide due to its high morbidity and mortality rates, leading to substantial economic losses. The clinical symptoms associated with various diarrhea pathogens often overlap, complicating accurate diagnosis; thus, there is an urgent need for rapid and precise diagnostic methods to improve prevention and treatment efforts. In this study, we developed a one-step multiplex reverse-transcription quantitative real-time polymerase chain reaction (mRT-qPCR) that enables the simultaneous detection of three key viral pathogens responsible for calf diarrhea: bovine kobuvirus (BKoV), bovine astrovirus (BoAstV), and bovine torovirus (BToV). However, development of accurate and rapid methods to distinguish these three viruses is helpful for the early detection, disease surveillance, and control of viral calf diarrhea. Specific primers and minor groove binder (MGB)-based probes were designed targeting the 3D region of BKoV, ORF1 region of BoAstV, and N region of BToV. The sensitivity, specificity, and reproducibility ability were evaluated for the mRT-qPCR. Further, 80 bovine fecal samples were subjected to the mRT-qPCR, and the results were verified using conventional reverse-transcription PCR (RT-PCR) or PCR methods and sequencing methods. This novel method demonstrated high sensitivity and specificity,achieving a detection limit of 24 copies/mL for each pathogen. Furthermore, the assay exhibited excellent reproducibility, with coefficients of variation below 1.5%, a strong linear correlation (R2 > 0.996), and an amplification efficiency between 90% and 110%. Validation with 80 clinical samples from both diarrheic and non-diarrheic cattle across four farms in Shanghai showed a high degree of concordance with RT-PCR, with positive detection rates for BKoV, BoAstV, and BToV at 28.75%, 8.75%, and 3.75%, respectively, highlighting the predominance of BKoV and BoAstV. Notably, this study represents the first identification of BKoV, BoAstV, and BToV in the Shanghai region. The mRT-qPCR is a robust, rapid, and simple tool for identifying viral pathogens associated with calf diarrhea, facilitating the development of effective prevention and control measures that are vital for the future sustainability of the cattle industry.

Accelerating the diagnosis of Chinese cblC type MMA patients by multiplex PCR sequencing method.

CblC type methylmalonic aciduria (cblC disease) is the most common inborn error of vitamin B12 metabolism and due to mutations in the MMACHC gene. The earlier the diagnosis, the better the prognosis. Therefore, convenient and inexpensive detection method is needed. This study selected mutational hot-spot regions in the MMACHC gene which harbors more than 90% of mutant alleles responsible for cblC disease in China. Subsequently, a hot-spot regions multi-PCR Sanger sequencing method (HsRMSS) was designed. The accuracy and efficiency of HsRMSS was validated using samples from 20 cblC families with known MMACHC gene mutations and 50 healthy volunteers. In addition, patients' clinical phenotypes and molecular genetic features were analyzed. A total of 16 different mutations were identified in 20 cblC families. Among them, the most common mutations were c.609 G>A (26/80, 32.5%), c.567dupT (10/80, 12.5%), c.80A>G (8/80, 10.0%), c.658_660delAAG (8/80, 10.0%) and c.394C>T (6/80, 7.5%), which accounted for over 70% of disease alleles. The HsRMSS results were the same as the results using the whole exon sequencing, with a coincidence rate of 100%. The HsRMSS targeting the mutational hot-spots of MMACHC gene could be a promising tool to accurately and rapidly diagnose cblC disease in China. This study reported the development and validation of a hot-spot regions multi-PCR Sanger sequencing method for targeting hotspots which harbor most of the common MMACHC gene mutations reported in Chinese patients with cblC disease. The approach could have a potential clinical application as a rapid diagnosis and screening tool for suspected children with cblC type MMA and population carrier, owing to its high throughput, low cost, and high sensitivity and specificity.

EVALUATION OF GRA6 AS GENETIC MARKER FOR DETERMINING TOXOPLASMA GONDII GENOTYPE IN THE CEREBROSPINAL FLUID OF HIV/AIDS PATIENTS WITH TOXOPLASMIC ENCEPHALITIS.

Toxoplasmic encephalitis is a severe manifestation of Toxoplasma gondii infection, with potentially fatal outcomes, particularly among immunocompromised patients. Clinical manifestation of this infection is associated with a specific genotype of T. gondii, requiring the use of genetic marker for genotype determination. This study critically evaluated the application of GRA6 gene as genetic marker for genotyping T. gondii in cerebrospinal fluid (CSF) samples from HIV/AIDS patients diagnosed with Toxoplasmic encephalitis. The study analyzed 69 CSF samples from HIV/AIDS patients with Toxoplasmic encephalitis. These samples tested positive for Toxoplasma IgG serology and SAG2 PCR, while GRA6 genotyping was conducted using PCR-sequencing methods. The results showed that GRA6 had potential for genotyping in positive control settings from culture cells. However, there was limited effectiveness in CSF samples from Toxoplasmic encephalitis patients. GRA6 had been proven effective as a genetic marker for the identification of T. gondii genotype among HIV/AIDS patients with Toxoplasmic encephalitis. However, the evaluation of GRA6 showed more effectiveness in cultured cells compared to direct clinical samples, such as cerebrospinal fluid obtained from HIV/AIDS patients with Toxoplasmic encephalitis.

<b>Isolation and identification of the H9N2 virus that caused the decline in poultry egg production in Indonesia from 2011 to 2018 using the RT–PCR-sequencing method</b>

Background: The Influenza A Virus subtype H9 is enzootic in portions of North and Central Africa, the Middle East, and Asia, where it significantly reduces the chicken industry's profitability. The H9N2 virus was reported in Indonesia in late 2016 causing decreased egg production and increased mortality. Research on the H9N2 virus is very important as an early warning of reassortment with potentially pandemic viruses such as the HPAI virus subtype H5N1. Aim: This study is a tracking study aimed at isolating and identifying the H9N2 virus in laying hens from samples from 2011-2018 in Indonesia. Methods: Sequencing was performed on 2949 samples obtained from tracheal and cloacal swabs after they were examined for the H9N2 virus using real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). Results: One positive sample came from West Java in 2017. The results of phylogenetic analysis revealed that this virus belongs to the China-Vietnam-Indonesia (CVI) lineage and is related to the A/chicken/West Java/VSN791/2017(H9N2) and A/chicken/Viet Nam/QN-2576/2015(H9N2) viruses. Conclusion: As an early warning system for AI outbreaks, active surveillance is still required to track the progress of LPAI viruses like H9N2.

Prevalence, genotyping and phylogenetic analysis of HPV infecting Palestinian women

BackgroundHuman Papillomavirus (HPV) is a significant global public health concern due to its association with cervical, other anogenital, and oropharyngeal cancers. This study aimed to evaluate the prevalence and the phylogenetic relationships of HPV among Palestinian in order to inform public health strategies.MethodsA cross-sectional study was conducted between September 2023 and April 2024 involving 379 Palestinian women over the age of 18 from 11 governorates in the West Bank. Cervical swabs were collected and analyzed using nested PCR and Sanger sequencing methods to detect and genotype HPV. The study also included phylogenetic analysis to understand the genetic relationships between HPV strains.ResultsThe overall HPV prevalence was 14.5%. The highest prevalence was observed in the 20–29 age group (19.6%), the Middle region of the West Bank (19.0%), and lower educational attainment. Genotyping revealed a diverse distribution of HPV types, with HPV 11 and HPV 6 being the most common low-risk types, while HPV 16 was the most common high-risk type. About 21.8% of the detected strains were high-risk strains. Phylogenetic analysis indicated significant regional clustering of HPV strains.ConclusionThe study highlights the need for targeted public health interventions, including vaccination and regular screening, particularly for younger women and those with lower educational attainment. Continued surveillance and research are essential to reduce the burden of HPV-related diseases in the West Bank, Palestine.

AAV vector-derived elements integrate into Cas9-generated double-strand breaks and disrupt gene transcription

AAV vector-derived elements integrate into Cas9-generated double-strand breaks and disrupt gene transcription

The Role of FLT3-ITD Mutation, PI3K/AKT Pathway, and Leukemia Stem Cells in D3A7 Induction therapy - the Outcomes of Adult Indonesian Patients with Acute Myeloid Leukemia.

This cohort study aimed to examine the impact of the FLT3-ITD mutation on the downstream signaling pathway of PI3K/AKT pathway, the percentage of leukemia stem cells, and the survival of patients receiving D3A7 induction therapy. Bone marrow mononuclear cells were collected from 20 adult AML patients who had completed D3A7 induction therapy at Cipto Mangunkusumo National General Hospital and Dharmais Cancer Hospital. FLT3-ITD gene mutation was examined by the PCR-sequencing method. Expression of phosphorylated PI3K and AKT was detected using the sandwich ELISA method. Flow cytometry was used for detecting the number of apoptosis and proliferation cells, and biomarkers of leukemia stem cells. The expression levels of PI3K and AKT proteins were higher in FLT3-ITD, both in the mutant group compared to the non-mutation group, and in the patient group with treatment failure outcomes compared to the patient group with treatment response. The percentage of the leukemia stem cell population did not differ significantly between the FLT3-ITD mutation group and the wild type group, and between the treatment failure outcome group and the response outcome group. This study presents the important role of FLT3-ITD mutation via its downstream signaling (PI3K/AKT) in the outcome of D3A7 induction therapy. The FLT3-ITD mutation plays an important role in the 12-month survival of AML patients after D3A7 therapy. However, the outcome of D3A7 therapy and FLT3-ITD mutation were not associated with leukemia stem cells.

Molecular identification and genotyping of Blastocystis spp. in children with clinical symptoms in southeast Iran using PCR-sequencing method

Blastocystis spp. is a zoonotic anaerobic parasite in the large intestine of humans and many vertebrates. It is mostly found in people who are in contact with animals. Current study aimed at identifying the prevalence of Blastocystis spp. and its common genotypes in children with clinical symptoms of diarrhea in the city of Zahedan, Southeast of Iran. A cross-sectional descriptive study was conducted on 60 children under ten years of age with gastrointestinal symptoms, especially diarrhea. After sample collection, stool samples were subjected to direct stool testing for initial diagnosis. After microscopic diagnosis, DNA extraction and Polymerase chain reaction (PCR) test with small subunit ribosomal RNA (SSU rRNA) gene target were performed, and PCR products were purified and sequenced. Nucleotide sequences were revised using Chromas biotechnology software version 2.4 and CLC genomic work bench software 11. Nucleotide sequences were aligned using BLAST database and compared with reference genotypes of Blastocystis spp. in gene bank. Genotyping of sequences was done using CLC genomic work bench software 11 and phylogenetic tree drawing was done using MEGA7 software with Neighbor-Joining statistical method applying Kimura 2-parameter method. Out of 60 examined cases, 5 children (8.33%) were positive by direct microscopic and PCR tests, where a 500 (479) bp fragment in the SSU-rRNA target was detected. Genetic analysis identified 4 subtypes, including subtypes 1, 2, 3, and 5. The percentage of nucleotide identity with the sequences in the gene bank was found to be between 93 and 100%. Considering the presence of subtypes 3 and 5 in the study and the evidence of their zoonotic, examining the parasite dynamics and epidemiological principles can be effective in the control strategy.

Modifiable risk factors, oxidative stress markers, and SOD2 rs4880 SNP in coronary artery disease: an association study.

Coronary artery disease (CAD) has been linked to single nucleotide polymorphism (SNP) in superoxide dismutase 2 (SOD 2) gene. Additionally, several modifiable risk factors are also known to influence the CAD risk. To investigate the association between selected modifiable risk factors and oxidative stress markers with the SOD2 rs4880 SNP in CAD patients. A cohort of 150 angiographically confirmed CAD patients, and 100 control subjects in the same geographic area were enrolled. SOD levels and lipid peroxidation were assessed in the blood samples using standard protocols. The genotyping of the SOD2 gene was conducted through the PCR-sequencing method. This study indicated that CAD patients with the rs4880 SNP having heterozygous AG and mutated homozygous GG genotypes have increased oxidative stress, decreased SOD activity, and a positive association with CAD risk (OR 2.85) in comparison with control individuals. The investigation among CAD patients was then carried out based on modifiable risk factors. The risk factors selected were clinical characteristics, physical habits, nutritional status, and body mass index. In all the cases, MDA levels showed a positive association, and SOD activity showed a negative association with the selected polymorphism. The study suggests that the selected modifiable risk factors have an important role in the higher oxidative stress found in patients, which may lead to SOD2 polymorphism. It also suggests that the SOD2 locus can be identified as a marker gene for CAD susceptibility.

Mutation-Selected Amplification droplet digital PCR: A new single nucleotide variant detection assay for [formula omitted] mutant in tumor and plasma samples

Mutation-Selected Amplification droplet digital PCR: A new single nucleotide variant detection assay for [formula omitted] mutant in tumor and plasma samples

Correlation between Gene polymorphism levels of serum matrix metalloproteinases with cavitary features and pulmonary fibrosis of the Patient tuberculosis multi-drug resistance using high-resolution computerized tomography of the Thorax

Correlation between Gene polymorphism levels of serum matrix metalloproteinases with cavitary features and pulmonary fibrosis of the Patient tuberculosis multi-drug resistance using high-resolution computerized tomography of the Thorax

The Prognostic Value of Somatic Mutations of Epigenetic Regulation Genes in Acute Myeloid Leukemias in Real-World Clinical Practice: Results of an Observational Non-Interventional Prospective Interregional Study

Aim. To assess the rate of DNMT3A, IDH1, IDH2, and ASXL1 gene mutations and their effect on the prognosis both as isolated findings and in combination with well-known chromosomal aberrations and gene mutations in newly diagnosed acute myeloid leukemia (AML) patients from some regions of the Russian Federation.
 Materials & Methods. The study enrolled 83 patients with newly diagnosed AML from 22 regions of the Russian Federation, who underwent molecular genetic examination for detecting IDH1 (R132), IDH2 (R140), ASXL1, and DNMT3A gene mutations with droplet digital PCR and Sanger sequencing methods.
 Results. The mutation rate in DNMT3A was 16.7 %, in IDH1 (R132) it was 6 %, in IDH2 (R140) it was 9.6 %, and in ASXL1 it was 6 %. The R140 mutation in IDH2 correlated with the older age of patients. The mutations in IDH1 (R132), IDH2 (R140), and DNMT3A showed a significant association with mutated NPM1. The mutations in IDH1 (R132), IDH2 (R140) were reported to occur significantly more often in patients with normal karyotype. The IDH1 (R132) and IDH2 (R140) mutations appeared to have a favorable effect on AML prognosis, which is most likely to be associated with a high rate of their compatibility with NPM1 mutation. The mutated type of DNMT3A had a negative effect on overall survival of patients with NPM1 mutation. The mutation in ASXL1 also appeared to be an unfavorable prognostic factor for overall survival of patients with wild type NPM1.
 Conclusion. A high rate of mutation occurrence in epigenetic regulation genes as well as the prognostic potential of these mutations in AML necessitate the need for determining the mutation status of DNMT3A, IDH1, IDH2, and ASXL1 in the context of primary diagnosis in real-world clinical practice.

Molecular biology techniques for assessing the loss of HLA heterozygosity after allogeneic hematopoietic stem cell transplantation in children with acute leukemia

According to several observations, up to a third of post-transplant relapses in childhood acute leukemia are associated with the loss of heterozygosity of the major histocompatibility complex (HLA). Furthermore, the inefficacy of the graft-versus-leukemia reaction, as evidenced by the lack of therapeutic effect from the infusion of donor lymphocytes, indicates the need for timely detection of this marker to change the treatment strategy in the post-transplant period. To detect the loss of HLA heterozygosity, the method using the commercial KMR-HLA system and analysis using next-generation sequencing (NGS), as well as the method based on the analysis of highly polymorphic STR and VNTR markers located in the HLA loci region on the short arm of chromosome 6, are widely used. The primary objective of our study was to compare the informativeness of these approaches in diagnosing HLA heterozygosity loss in children during the post-transplant period. The obtained data on the frequency of detecting HLA heterozygosity loss were comparable to the literature data and constituted 23 % of cases of post-transplant relapse of B-cell acute lymphoblastic leukemia, 33 % of cases of T-cell acute lymphoblastic leukemia, and 23% of cases of acute myeloid leukemia. We also demonstrated that the method based on STR marker analysis has sensitivity comparable to allele-specific PCR and NGS sequencing methods. Meanwhile, preliminary sorting of the blast population increases the sensitivity of STR analysis and can be recommended in routine practice.

Fish species mislabelling in China: evidence from a survey of frozen, dried, and surimi-based fish products marketed as cod-related species

‘Cod’-related species are among the most appreciated marine fish resources around the world, but are also prone to species mislabelling. In the present study, a total of 76 frozen, dried, and surimi-based fish products, sold as ‘Cod’ (59 products), ‘Atlantic authentic Cod’ (11 products), and ‘Authentic Cod’ (6 products), were collected in China. A species-specific LAMP (loop-mediated isothermal amplification) method was used to screen for the presence of Atlantic cod (Gadus morhua), Pacific cod (G. macrocephalus), Alaska pollock (G. chalcogrammus), Southern hake (Merluccius australis), which was cross-confirmed using real-time PCR and DNA sequencing methods. The results highlighted the greatest species diversity for ‘Cod’ products, and the identified species were from nine different families. It appears that the practice of assigning a specific type or category of species to the common name ‘Cod’ has not been widely advocated, and the misuse of this ambiguous common name has been a common practice for species adulteration, negatively impacting consumers’ rights and marine conservation. To rebuild consumers’ confidence, retail fish suppliers have differentiated their products by adding specific qualifiers in front of the common name ‘Cod’ on the label, such as ‘Authentic cod’ and ‘Atlantic authentic cod’. The endeavour is highly meaningful, since Gadus morhua was identified as the species for a significant majority of ‘Atlantic authentic cod’ and ‘Authentic cod’ products (64.7%, 11/17), with the remaining six products identified as Alaskan pollock (G. chalcogrammus), Pacific cod (G. macrocephalus) and North Pacific hake (Merluccius productus). Despite the positive effort to reverse species mislabelling from retail on-line fish suppliers, a standardized fish nomenclature stipulated by the responsible authorities remains crucial for enhancing transparency and continuing to reduce species mislabelling.

The complete mitochondrial genome of Semiothisa cinerearia Bremer & Grey 1853 (Lepidoptera: Geometridae: Ennominae) and its phylogenetic analysis

Semiothisa cinerearia Bremer & Grey, 1853 belongs to the lepidopteran family Geometridae, subfamily Ennominae. We sequenced the complete mitochondrial genome of S. cinerearia by PCR and Sanger sequencing method. The mitochondrial genome of S. cinerearia is 15,523 bp in length and contains a typical set of 37 genes with ‘MIQ’ type gene arrangement and a 394 bp AT-rich regions. Except for cox1 using CGA as initiation codon, all protein-coding genes (PCGs) start with ATN codons and except for nad2 and nad4l using TAG as termination codon, all PCGs terminated with TAA codon. A phylogenetic tree including 39 genus of subfamily Ennominae was first reconstructed based on the mitochondrial genome sequences with nucleotide substitution model GTR + G + I, which showed that the genera Amraica, Jankowskia, and Ectropis are not monophyletic and S. cinerearia and Macaria notata are classified together.