Method For Detection Of Genes Research Articles

Keratin 20 positive SDH-deficient renal cell carcinoma: a case report and literature review

This study aims to broaden the morphological scope of SDH-deficient renal cell carcinoma and to assist clinicians and pathologists in better understanding this entity to prevent misdiagnosis. This study used immunohistochemistry staining and the first-generation sequencing Sanger method for gene detection. It retrospectively analysed the clinical pathology, molecular characteristics, biological behaviour, and treatment information of one case of SDH-deficient renal cell carcinoma. The patient was a 57-year-old female with right back pain for more than 20 days and had no personal or family history of kidney tumours. In addition, the tumour cells had clear boundaries in morphology, and residual normal renal tubules could be seen around them. There were also ossification and adipose tissue around the tumour centre. The tumour cells were arranged in a glandular tubular and cord-like manner. Vacuolar and eosinophilic inclusion bodies could be observed in the cytoplasm. The nucleus was regular, the chromatin distribution was fine, and there were no obvious nucleoli. They were low-grade nuclei. In addition, no atypical mitosis or necrosis could been found. Furthermore, immunohistochemistry staining showed SDHB-negative and keratin 20 -positive tumour. Meanwhile, the first-generation sequencing also pointed out the presence of SDHB gene mutations in the tumour. After 12 months of follow-up, there was no evidence of disease recurrence in the patient. SDH-deficient renal cell carcinoma is a rare tumour associated with SDH gene germline mutations, and suspected cases should undergo SDHB immunohistochemistry staining. Most SDH-deficient renal cell carcinomas have a good prognosis, but undifferentiated cases require long-term follow-up.

Standardization and application of ARMS TaqMan real-time PCR for screening of folate metabolism genes in Han Chinese.

Folate has antioxidant properties, and low concentration in seminal plasma may be associated with increased DNA damage in sperm. Mutations of the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes, including MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), and MTRR A66G (rs1801394), can lead to decreased activity of the encoded folate metabolic enzymes, thereby affecting male reproduction. The current SNP detection methods commonly used in clinical practice have some shortcomings, such as long time-consuming, complex detection steps, or high cost. The purpose of this study was to establish a simple, time-saving, sensitive, accurate, and easy to clinical popularization method for folate metabolism gene detection. We combined ARMS-PCR with TaqMan fluorescent probe to establish an ARMS TaqMan real-time PCR detection method. According to the variation of rs1801131, rs1801133, and rs1801394, two specific primers (one wild type and one mutant) were designed. Mismatched nucleotides were introduced at the penultimate or third position to improve the specificity of the primer. Specific TaqMan probe was introduced to detect PCR products to improve the sensitivity of the method. The results showed that the sensitivity of ARMS TaqMan real-time PCR in SNP genotyping was 1ng, and the accuracy was 100%. A total of 249 clinical samples were detected by the established method, and the correlation between three SNPs and semen quality was analyzed. We found that individuals carrying the AG+GG genotype of rs1801394 had a lower risk of abnormal semen quality. In conclusion, we developed a highly sensitive, accurate, rapid, and easy to be popularized method for detecting SNPs of rs1801394, rs1801131, and rs1801133. ARMS TaqMan real-time PCR is a reliable SNP genotyping method in folate metabolism genes.

1199P Developing and systematically validating homologous recombination repair gene detection method based on next-generation sequencing

1199P Developing and systematically validating homologous recombination repair gene detection method based on next-generation sequencing

An Integrated Micro-Heating System for On-Chip Isothermal Amplification of African Swine Fever Virus Genes.

The loop-mediated isothermal amplification (LAMP) is widely used in the laboratory to facilitate rapid DNA or RNA detection with a streamlined operational process, whose properties are greatly dependent on the uniformity and rise rate of temperature in the reaction chambers and the design of the primers. This paper introduces a planar micro-heater equipped with an embedded micro-temperature sensor to realize temperature tunability at a low energy cost. Moreover, a control system, based on the Wheatstone bridge and proportional, integral, and derivative (PID) control, is designed to measure and adjust the temperature of the micro-heater. The maximum temperature rise rate of the designed micro-heater is ≈8°Cs-1, and it only takes ≈60s to reach the target temperature. Furthermore, a designed plasmid, containing the B646L gene of African Swine Fever Virus (ASFV), and a set of specific primers, are used to combine with the designed micro-heating system to implement the LAMP reaction. Finally, the lateral flow assay is used to interpret the amplification results visually. This method can achieve highly sensitive and efficient detection of ASFV within 40min. The sensitivity of this on-chip gene detection method is 8.4 copies per reaction, holding great potential for applications in DNA and RNA amplification.

Detection of hemophilia A genetic variants using third-generation long-read sequencing

BackgroundHemophilia A (HA) is an X-linked recessive genetic disorder caused by pathogenic variations of the factor VIII −encoding gene, F8 gene. Due to the large size and diverse types of variations in the F8 gene, causative mutations in F8 cannot be simultaneously detected in one step by traditional molecular analysis, and genetic molecular diagnosis and prenatal screening of HA still face significant difficulties and challenges in clinical practice. Therefore, we aimed to develop and validate an efficient, accurate, and time-saving method for the genetic detection of HA. MethodsA comprehensive analysis of hemophilia A (CAHEA) method based on long-range PCR and long-read sequencing (LRS) was used to detect F8 gene mutations in 14 clinical HA samples. The LRS results were compared with those of the conventional methods to evaluate the accuracy and sensitivity of the proposed approach. ResultsThe CAHEA method successfully identified 14 F8 variants in all probands, including 3 small insertion deletions, 4 single nucleotide variants, and 7 intron 22 inversions in a “one-step” manner, of which 2 small deletions have not been reported previously. Moreover, this method provided an opportunity to analyze the mechanism of rearrangement and the pathogenicity of F8 variants. The LRS results were validated and found to be in 100% agreement with those obtained using the conventional method. ConclusionOur proposed LRS-based F8 gene detection method is an accurate and reproducible genetic screening and diagnostic method with significant clinical value. It provides efficient, comprehensive, and accurate genetic screening and diagnostic services for individuals at high risk of HA as well as for premarital and prenatal populations.

Dual Gene Detection of H5N1 Avian Influenza Virus Based on Dual RT-RPA.

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10-7 ng/μL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 (p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus.

Application of the TaqMan ARMS-PCR Approach for Genotyping Drug-Induced Hearing Loss Using Dried Blood Samples.

A single nucleotide variant in mitochondrial DNA (mtDNA) 1555A>G is associated with drug-induced hearing loss. For the 1555A>G mutation site, 1555A wild-type and 1555G mutant-type plasmids were constructed, respectively. In this study, a PCR method based on the TaqMan amplification refractory mutation system was proposed to detect mtDNA 1555A>G. A common upstream primer, a common TaqMan probe, and two downstream allele-specific primers with mismatched bases were designed. One-step amplification and detection of the wild-type and mutant type at the 1555 site were realized for the deafness-related gene through two reactions. Based on this detection method, the minimum detection limit of the wild-type and mutant type detection systems for plasmids was 50 copies/μL. The minimum sensitivity for the detection of nucleic acids in real dried blood spot (DBS) samples was 0.1 ng/μL. In the normal DBS DNA sample, the detection limit of the mutation abundance reached 0.78%. The specificity of the detection method was 100%, and the coefficient of variation was less than 3.36%. This approach was validated using clinical DNA extracted from 113 DBS samples of newborns. Additionally, it showed 100% agreement with bi-directional Sanger sequencing. It can be used as an optional method for the clinical detection of deafness-related genes.

Establishment and application of recombinase polymerase amplification combined with a lateral flow dipstick for the detection of mcr-1 in uncultured clinical samples

The rapid dissemination of the mcr-1 gene via plasmid-mediated transfer has raised concerns regarding the efficacy of colistin as a last-resort treatment for multidrug-resistant Gram-negative bacterial infections. Current mcr-1 gene detection methods mainly focus on cultured bacteria, which is a complex and time-consuming process requiring skilled personnel, making it unsuitable for field analysis. A rapid detection technique combining recombinase polymerase amplification with a lateral flow dipstick targeting uncultured clinical samples was developed. This new method targeting the mcr-1 gene region (23 232-23 642 bp, no. KP347127.1) achieved a low detection limit of 10 copies/μL. The whole process was carried out with high specificity and was completed within 20 min. The evaluation assay was conducted using 45 human faecal samples; 16 strains yielded a 98% accuracy, closely matching antimicrobial susceptibility outcomes. The novel method integrates nucleic acid extraction, isothermal amplification, and a test assay, suggesting the potential for timely colistin resistance surveillance in frontline disease control and healthcare settings, supporting future prevention and clinical standardization efforts.

Evaluating spatially variable gene detection methods for spatial transcriptomics data

BackgroundThe identification of genes that vary across spatial domains in tissues and cells is an essential step for spatial transcriptomics data analysis. Given the critical role it serves for downstream data interpretations, various methods for detecting spatially variable genes (SVGs) have been proposed. However, the lack of benchmarking complicates the selection of a suitable method.ResultsHere we systematically evaluate a panel of popular SVG detection methods on a large collection of spatial transcriptomics datasets, covering various tissue types, biotechnologies, and spatial resolutions. We address questions including whether different methods select a similar set of SVGs, how reliable is the reported statistical significance from each method, how accurate and robust is each method in terms of SVG detection, and how well the selected SVGs perform in downstream applications such as clustering of spatial domains. Besides these, practical considerations such as computational time and memory usage are also crucial for deciding which method to use.ConclusionsOur study evaluates the performance of each method from multiple aspects and highlights the discrepancy among different methods when calling statistically significant SVGs across diverse datasets. Overall, our work provides useful considerations for choosing methods for identifying SVGs and serves as a key reference for the future development of related methods.

Simultaneous detection of AFB1 and aflD gene by "Y" shaped aptamer fluorescent biosensor based on double quantum dots.

The developed method for simultaneous detection of aflatoxin B1 (AFB1) and aflD genes can effectively monitor from the source and reduce the safety problems and economic losses caused by the production of aflatoxin, which can be of great significance for food safety regulations. In this paper, we constructed a sensitive and convenient fluorescent biosensor to detect AFB1 and aflD genes simultaneously based on fluorescence resonance energy transfer (FRET) between quantum dots (QDs) and a black hole quenching agent. A stable "Y" shaped aptasensor was employed as the detection platform and a double quantum dot labeled DNA fragment was utilized to be the sensing element in this work. When the targets of AFB1 and aflD genes were presented in the solution, the aptamer in the "Y" shaped probe is specifically recognized by the target. At this time, both Si-carbon quantum dots (Si-CDs) and CdTe QDs are far away from the BHQ1 and BHQ3 to recover the fluorescence. The linear range of the prepared fluorescence simultaneous detection method was as wide as 0.5-500ng·mL-1 with detection lines of 0.64ng·mL-1 for AFB1 and 0.5-500nM with detection lines of 0.75nM for aflD genes (3σ/k). This fabricated fluorescent biosensor was further validated in real rice flour and corn flour samples, which also achieved good results. The recoveries were calculated by comparing the known and found amounts of AFB1 which ranged from 88.4 to approximately 115.32% in the rice flour samples and 90.7 ~ 102.58% in the corn flour samples. The recoveries of aflD genes ranged from 84.32 to approximately 109.3% in the rice flour samples and 89.48 ~ 100.99% in the corn flour samples. Therefore, the proposed biosensor can significantly improve food safety and quality control through a simple, fast, and sensitive agricultural product monitoring and detection system.

Analysis of gene variation and long-term follow-up in children with phenylalanine hydroxylase deficiency diagnosed by newborn screening.

To retrospectively analyze the variation and characteristics of phenylalanine hydroxylase (PAH) gene, and to observe the long-term treatment effect and follow-up of newborns with PAH deficiency. Clinical data, treatment and follow-up results of 198 patients with PAH deficiency diagnosed by newborn screening in Jinan from 1996 to 2021 were collected. The genetic analysis of 55 patients with PAH deficiency diagnosed by newborn screening in Jinan and 213 patients referred from the surrounding areas of Jinan were summarized. Gene variations were checked by a customized Panel gene detection method. Blood phenylalanine-concentration and physical development indicators including height and weight were regularly monitored. Intellectual development was assessed using a neuropsychological development scale for patients aged 0-6 years and academic performance, and brain injury in patients was assessed using brain magnetic resonance imaging. c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G variations were common in PAH gene. The genotype of c.158G>A variation is compound heterozygous variation, with mainly a mild hyperpheny-lalaninemia. 168 patients with PAH deficiency who were followed-up regularly had normal physical development without dwarfism or malnutrition. Among the 33 preschool patients who underwent mental development assessment, 2 were mentally retarded and the initial treatment age was older than 6 months. Nine patients with an average age of (17.13±2.42) years completed brain magnetic resonance imaging, one case was normal, and 8 cases were abnormal. There were patchy or patchy hyperintense foci near the bilateral lateral ventricles on T2WI, and the intellectual development was normal. Compared with the other eight patients, the blood phenylalanine concentration of the normal child was better and stably controlled within the ideal range. c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G variations were common in PAH gene. After standardized treatment, most patients with PAH deficiency diagnosed by screening can obtain normal growth and intellectual development in adolescence, but there are different degrees of organic lesions in the cerebral white matter.

Analysis of Klebsiella Pneumonia and Resistance Genes Coding and Carbapenemase Production in National Referral Hospital Wastewater

Hospitals, as a source of transmission and spread of bacteria carrying antibiotic-resistant genes (ARGs) as well as a high source of antibiotic compounds, are a significant reservoir of antibiotic-resistant gene transfer that causes bacteria to develop multidrug resistance (MDR). Klebsiella pneumonia is a bacterium often found in wastewater with a high level of Extended Spectrum β-lactamase (ESBL) and carbapenemase resistance. This study aimed to determine Klebsiella pneumonia and resistance genes encoding ESBL and carbapenemase in hospital wastewater to obtain primary Antimicrobial Resistance (AMR) data in the environment, which was first conducted in Indonesia. The resistance gene detection method was developed using High Throughput Real-Time and conventional Real-Time (HT-RT PCR). The data obtained were relative abundance and copy number. The results showed that blaTEM and blaNDM were the highest ARGs. Conventional Real-Time PCR results showed better sensitivity in detecting K. pneumoniae and ARGs than HT-RT PCR. The detection of K. pneumoniae and ARGs in RSCM wastewater in Indonesia indicates the need to improve the handling of RSCM WWTP to monitor the number of microbial resistances to antibiotics.

Optimization of deoxyribonucleic acid extraction and polymerase chain reaction methods for gene detection of Toxoplasma gondii in goat milk

Abstract. Fatmawati M, Suwanti LT, Mufasirin, Subekti DT, Ekawasti F, Lastuti NDR, Lamid M, Suprihati E, Effendi MH, Al-Arif A. 2023. Optimization of deoxyribonucleic acid extraction and polymerase chain reaction methods for gene detection of Toxoplasma gondii in goat milk. Biodiversitas 24: 5905-5911. Dairy goats (Capra aegagrus hircus) are intermediate hosts that transmit Toxoplasma gondii to humans through goat milk consumption. T. gondii can invade mammary glands and secrete into milk. The method of detecting gene of the T. gondii in milk is through a Polymerase Chain Reaction (PCR) test based on specific genes. The objective of this study was to compare the efficacy methods of the T. gondii molecular assay from the extraction, master mix for PCR assay, and primer that is possible to amplify the gene of T. gondii in goat milk for PCR assay. The research method for purification used flotation using concentrated sugar. This research compared the extraction kit from Kit Extraction 1 (K1), Kit Extraction 2 (K2), and Kit Extraction 3 (K3) with the extraction method listed in the brochure. Meanwhile, for the PCR assay, three types of master mix were used, including Master Mix 1 (M1), Master Mix 2 (M2), and Master Mix 3 (M3). The specific genes used included primers B1, ROP, GRA 1, GRA 7, BAG 1, and P30. Tris borate EDTA (TBE) or Phosphate Buffer Saline Solution (PBS) as buffer milk samples gave the same PCR test results. The results show that the principle of a flotation method test using PBS can be used to prepare the milk sample. The research showed that extraction kit 1 (K1) and extraction kit 2 (K2) gave positive results for the positive control. However, in terms of price, K2 is cheaper than K1. So, to optimize DNA extraction, K2 can be used. The master mix that can be used for PCR testing of milk samples is M1 because M1 gives positive results for positive controls and negative results for negative controls. Primers for optimal results used GRA 7 and B1 with denaturation temperature of 94°C, annealing at 56°C, and an extension of 73°C 33. This research concluded that the type of DNA extraction kit affects the PCR test results for detecting T. gondii in goat milk. It is necessary to optimize the type of master mix to be used in PCR testing and primers capable of amplifying the T. gondii gene.

Nucleic Acid Amplification Free-QCM-DNA Biosensor for Burkholderia pseudomallei Detection.

Burkholderia pseudomallei is a gram-negative bacterium that causes the infectious disease melioidosis, a disease that can still be fatal despite appropriate treatment. The bacterium contains the gene clusters for the type III secretion system (TTSS), which are essential for its pathogenicity. This gene was often employed for accurate diagnosis through the laborious process of gene amplification. This work intends to develop a quartz crystal microbalance (QCM)-based TTSS gene detection method without gene amplification approaches to simplify the diagnosis process. In this study, it was demonstrated that a 540bp sequence flanked by BglI restriction sites within the TTSS1 on the B. pseudomallei genome is an effective target for specific detection of the bacteria. After cultivation and genome extraction, the bacteria can be detected by digesting its genome with BglI in which the TTSS1 fragment is detected by a QCM-DNA biosensor, eliminating the need for nucleic acid amplification. A specific probe designed to bind to the TTSSI fragment was utilized as the receptor on the QCM-DNA biosensor which provided the ability to detect the fragment. The limit of detection of the QCM-DNA biosensor was 0.4µM of the synthetic DNA target oligonucleotide. The system was also capable of specifically detecting the BglI digested-DNA fragment of B. pseudomallei species with significantly higher signal than B. thailandensis. This study provides evidence for an effective QCM-DNA biosensor that can identify B. pseudomallei without the need for nucleic acid amplification.

A gold nanoparticle colorimetric probe-based biosensor for rapid detection of VIM-2 and IMP-1 metallo-beta lactamase genes

Despite actions to reduce the prevalence of antibiotic resistance, antibiotic resistance remains a major threat in the treatment and control of infections. Therefore, the development of rapid diagnostic methods plays a significant role in the detection of antibiotic resistance genes and the management of hospital infections. This study aimed to design and develop a gold nanoparticle colorimetric probe-based biosensor was developed for faster and more accurate detection of VIM-2 and IMP-1 Metallo-Beta-Lactamases (MBLs) genes in different clinical samples. After identifying 248 clinical isolates of bacteria with standard biochemical methods and evaluation of antibiotic resistance and identifying MBL-producing strains, PCR method was carried out for detecting VIM-2 and IMP-1 genes as a gold standard. Synthesis of AuNPs were done by citrate reduction method and AuNP -IMP-1 biosensor was used for detecting IMP-1 gene, after functionalization of thiol modified oligonucleotides. AuNP biosensor and IMP-1 PCR were compared in terms of detection indices. Method PCR was defined as the gold standard. Bacteria examined, 87 isolates were resistant to carbapenems. Out of the 87 carbapenem-resistant isolates, 85 (34.2%) were phenotypically positive for MBLs. Also, 7 isolates had IMP-1 gene, but none of them carried VIM-2 gene. The AuNP biosensor had 100% sensitivity, specificity, PPV and NPV, respectively. The final detection limit of IMP-1 genomic DNA (LOD) by PCR and AuNP-IMP-1 biosensor technique was 0.1 fg/μl (2.5 fg/25 reactions) and 0.001 fg/μl (0.025 fg/25 reaction), respectively. According to the promising results of Diagnostic indices, AuNP biosensor is a more efficient and accurate method for direct and indirect detection of antibiotic resistance genes in clinical samples.

Clinical utility of HPV typing and quantification combined with PAX1/ZNF582 methylation detection in accurate cervical cancer screening.

This article aims at exploring the clinical value of high-risk human papillomavirus (HPV) positive and paired boxed 1 (PAXI)/zinc finger protein 582 (ZNF582) gene methylation shunt as a new approach for accurate cervical cancer screening. Selecting 115 patients were treated in the Cervical Department of Xuzhou Matemal and Child Health Hospital from October 2018-October 2020. All patients underwent cervical exfoliated cell thinprep cytologic test (TCT) detection, HPV typing quantitative detection, and PAX1/ZNF582 gene methylation level Detection. Taking the biopsy pathological diagnosis under colposcopy as the gold standard, analyzing the test results statistically, and the sensitivity, specificity, and accuracy of the three screening methods alone and combined screening schemes were compared. Comparison of the three methods of cervical exfoliated cell TCT, HPV typing and quantification, and PAX1/ZNF582 methylation gene detection showed that the gene detection method has the highest specificity, 97.30%; The HPV typing quantitative detection has the highest sensitivity, 89.71%, but its specificity is poor; and the PAX1/ZNF582 gene detection has the highest accuracy. For patients with high-grade cervical lesions and cervical cancer, PAX1/ZNF582 gene methylation level can be used as an important biomarker for the diagnosis and classification of cervical cancer. PAX1/ZNF582 methylation gene detection is effective in high-grade cervical lesions and cervical cancer. Screening has high clinical value and can become a new way of accurate cervical cancer screening.

Advances and Trends in miRNA Analysis Using DNAzyme-Based Biosensors.

miRNAs are endogenous small, non-coding RNA molecules that function in post-transcriptional regulation of gene expression. Because miRNA plays a pivotal role in maintaining the intracellular environment, and abnormal expression has been found in many cancer diseases, detection of miRNA as a biomarker is important for early diagnosis of disease and study of miRNA function. However, because miRNA is present in extremely low concentrations in cells and many types of miRNAs with similar sequences are mixed, traditional gene detection methods are not suitable for miRNA detection. Therefore, in order to overcome this limitation, a signal amplification process is essential for high sensitivity. In particular, enzyme-free signal amplification systems such as DNAzyme systems have been developed for miRNA analysis with high specificity. DNAzymes have the advantage of being more stable in the physiological environment than enzymes, easy to chemically synthesize, and biocompatible. In this review, we summarize and introduce the methods using DNAzyme-based biosensors, especially with regard to various signal amplification methods for high sensitivity and strategies for improving detection specificity. We also discuss the current challenges and trends of these DNAzyme-based biosensors.

Multiplex solid-phase RPA coupled CRISPR-based visual detection of SARS-CoV-2

The COVID-19 pandemic has presented a significant challenge to the world's public health and led to over 6.9 million deaths reported to date. A rapid, sensitive, and cost-effective point-of-care virus detection device is essential for the control and surveillance of the contagious severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. The study presented here aimed to demonstrate a solid-phase isothermal recombinase polymerase amplification coupled CRISPR-based (spRPA-CRISPR) assay for on-chip multiplexed, sensitive and visual COVID-19 DNA detection. The assay targets the SARS-CoV-2 structure protein encoded genomes and can simultaneously detect two specific genes without cross-interaction. The amplified target sequences were immobilized on the one-pot device surface and detected using the mixed Cas12a-crRNA collateral cleavage of reporter-released fluorescent signal when specific genes were recognized. The endpoint signal can be directly visualized for rapid detection of COVID-19. The system was tested with samples of a broad range of concentrations (20 to 2 × 104 copies) and showed analytical sensitivity down to 20 copies per microliter. Furthermore, a low-cost blue LED flashlight (∼$12) was used to provide a visible SARS-CoV-2 detection signal of the spRPA-CRISPR assay which could be purchased online easily. Thus, our platform provides a sensitive and easy-to-read multiplexed gene detection method that can specifically identify low concentration genes.

NGS-based targeted sequencing identified six novel variants in patients with Duchenne/Becker muscular dystrophy from southwestern China

BackgroundAt present, Multiplex ligation-dependent probe amplification (MLPA) and exome sequencing are common gene detection methods in patients with Duchenne muscular dystrophy or Becker muscular dystrophy (DMD/BMD), but they can not cover the whole-genome sequence of the DMD gene. In this study, the whole genome capture of the DMD gene and next-generation sequencing (NGS) technology were used to detect the patients with DMD/BMD in Southwest China, to clarify the application value of this technology and further study the gene variant spectrum.MethodsFrom 2017 to 2020, 51 unrelated patients with DMD/BMD in southwestern China were clinically diagnosed at West China Second University Hospital of Sichuan University (Chengdu, China). The whole-genome of the DMD gene was captured from the peripheral blood of all patients, and next-generation sequencing was performed. Large copy number variants (CNVs) in the exon regions of the DMD gene were verified through MLPA, and small variations (such as single nucleotide variation and < 50 bp fragment insertions/deletions) were validated using Sanger sequencing.ResultsAmong the 51 patients, 49 (96.1% [49/51]) had pathogenic or likely pathogenic variants in the DMD gene. Among the 49 positive samples, 17 patients (34.7% [17/49]) had CNVs in the exon regions and 32 patients (65.3% [32/49]) had small variations. A total of six novel variants were identified: c.10916_10917del, c.1790T>A, c.1842del, c.5015del, c.5791_5792insCA, and exons 38–50 duplication.ConclusionsPathogenic or likely pathogenic variants of the DMD gene were detected in 49 patients (96.1% [49/51]), of which 6 variants (12.2% [6/49]) had not been previously reported. This study confirmed the value of NGS-based targeted sequencing for the DMD gene expanding the spectrum of variants in DMD, which may provide effective genetic counseling and prenatal diagnosis for families.

N-3 PUFA Promotes Ferroptosis in PCOS GCs by Inhibiting YAP1 through Activation of the Hippo Pathway.

Polycystic ovary syndrome (PCOS) is an endocrine disorder characterized by hyperandrogenemia with multiple suspended sinus follicles, thickened cortical tissue, and excessive proliferation of ovarian granulosa cells that severely affects the fertility and quality of life of women. The addition of n-3 PUFA to the diet may slightly reduce body weight and greatly alleviate disturbed blood hormone levels in PCOS mice. We treated KGN as a cell model for n-3 PUFA addition and showed that n-3 PUFA inhibited the proliferation of GCs and promoted ferroptosis in ovarian granulosa cells. We used CCK-8, fluorescence quantitative transmission electron microscopy experiments and ferroptosis marker gene detection and other methods. Furthermore, n-3 PUFA was found to promote YAP1 exocytosis by activating Hippo and weakening the cross-talk between YAP1 and Nrf2 by activating the Hippo signaling pathway. In this study, we found that n-3 PUFA inhibited the over proliferation of granulosa cells in ovarian follicles by activating Hippo, promoting YAP1 exocytosis, weakening the cross-talk between YAP1 and Nrf2, and ultimately activating the ferroptosis sensitivity of ovarian granulosa cells. We demonstrate that n-3 PUFA can alleviate the hormonal and estrous cycle disorder with PCOS by inhibiting the YAP1-Nrf2 crosstalk that suppresses over proliferating ovarian granulosa cells and promotes iron death in GCs. These findings reveal the molecular mechanisms by which n-3 PUFA attenuates PCOS and identify YAP1-Nrf2 as a potential therapeutic target for regulation granulosa cells in PCOS.