Fluorescent Polymerase Chain Reaction Research Articles

Accurate and reliable surface-enhanced Raman spectroscopy assay for early detection of SARS-CoV-2 RNA with exceptional sensitivity

This research proposes a highly sensitive and simple surface-enhanced Raman spectroscopy (SERS) assay for the detection of SARS-CoV-2 RNA using suitably designed probes specific for RdRp and N viral genes attached to a Raman marker. The sensitivity of the assay was optimized through precise adjustments to the conditions of immobilization and hybridization processes of the target RNA, including modifications to factors such as time and temperature. The assay achieved a remarkable sensitivity down to 58.39 copies/mL, comparable to or lower than the sensitivities reported for commercial fluorescent polymerase chain reaction (PCR) based methods. It has good selectivity in discriminating SARS-CoV-2 RNA against other respiratory viruses, respiratory syncytial virus (RSV), and influenza A virus. The reliability of the assay was validated by testing 24 clinical samples, including 12 positive samples with varying cycle threshold (Ct) values and 12 negative samples previously tested using real-time PCR. The assay consistently predicted true results that were in line with the PCR results for all samples. Furthermore, the assay demonstrated a notable limit of detection (LOD) of Ct (38 for RdRp gene and 37.5 for N-gene), indicating its capability to detect low concentrations of the target analyte and potentially facilitating early detection of the pathogen.

Copy number variation sequencing for the products of conception: What is the optimal testing strategy

BackgroundCopy number variation sequencing (CNV-seq) is crucial in prenatal diagnosis, but its limitations in detecting polyploidy, maternal cell contamination (MCC), and uniparental disomy (UPD) restrict its application in the analysis of products of conception (POCs). This study aimed to investigate an optimal genetic testing strategy for POCs in the era of CNV-seq. MethodsCNV-seq and quantitative fluorescent polymerase chain reaction (QF-PCR) were performed in all 4,211 spontaneous miscarriage cases. Different testing strategies were compared and the optimal testing strategies were proposed. ResultsOf the 4,211 cases, 2561 (60.82%) exhibited clinically significant chromosomal abnormalities. CNV-seq alone, without QF-PCR, might misdiagnose 311 (7.39%) cases, including 278 polyploidy, 13 UPD, and 20 MCC. In 20 MCC cases identified by QF-PCR, CNV-seq successfully pinpointed the cause of miscarriage in 13 cases. Furthermore, in cases where QF-PCR suggested polyploidy, CNV-seq improved the diagnostic accuracy in 54 (1.28%) hypo/hypertriploidy cases. After comparing four different strategies, the sequential approach (initiating with CNV-seq followed by QF-PCR if necessary) emerged as advantageous, reducing approximately 70% of the cost associated with QF-PCR while maintaining result accuracy. ConclusionsWe propose an initial CNV-seq followed by QF-PCR if needed—an efficient and cost-effective strategy for the genetic analysis of POCs.

Characteristics of reproductive tract infections caused by common pathogens among the outpatients of reproductive medicine center in Putian: retrospective study

BackgroundThis study aims to explore the infection and age distribution of Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Herpes simplex virus type II (HSV II) among the outpatients of Reproductive Medicine Center in Putian, Fujian Province to provide a clinical basis for the early diagnosis and treatment of various reproductive tract diseases and infertility in this region.MethodsA total of 1736 samples of secretions and exfoliated cervical cells were collected from the outpatients of the Reproductive Medicine Center of the Affiliated Hospital of Putian University from December 2021 to April 2023. The infections of UU, CT, NG and HSVII were detected by real-time fluorescence polymerase chain reaction (PCR), and the infection statuses of the patients with different genders, ages and diagnoses were analysed.ResultsAmong the 1736 patients, 611 were male and 1125 were female. The male patients had higher UU infection rate but lower HSV II infection rate than the female patients. No significant difference in CT and NG infection rates was observed between the genders. The CT infection rate gradually decreased with the increase in the age. The difference in UU, NG and HSV II infection rates among the different age groups was not statistically significant. For UU infection, the male infertile patients had the highest rate of 37.72% (172/456). Meanwhile, the differences in CT, NG and HSV II infection rates among the different diagnosis groups were not statistically significant. Among the male and female infertile patients, the CT infection rate was the highest in the 21–25 years of age group at 11.11% (2/18) and 9.47% (9/95), respectively. No statistically significant difference in UU, CT, NG and HSV II infection rates was observed among the different age groups of patients diagnosed in relation to the family planning guidance and between the male and female patients with other diagnoses results.ConclusionsThis study showed that UU was the most frequently identified pathogen in infertile men in Putian, Fujian Province. The CT infection rate was the highest in people under 20 years old, and the infection showed a tendency toward young individuals. Therefore, the publicity of sexual health knowledge must be strengthened, and the prevention and treatment of venereal diseases among young and middle-aged people must be improved. Moreover, the pathogen infection is related to infertility to a certain extent, which is conducive to clinical diagnosis and treatment.

Blood Chimerism in Fertile Female with 46, XY Karyotype Report

Introduction: The mammalian Y chromosome is a vital sex-determining factor in mammals by encoding gonadal genes. However, there are some discordant conditions with the XY female chromosome, which can lead to their inclusion in the disorders of sex development group.
 Case Report: The person under study is a 22-year-old woman for whom written consent was obtained, and all ethical procedures were followed. Herein, we report an extraordinarily fertile adolescent phenotypic mother with congenital XY abnormality who reared as female, spontaneous puberty with regular menstruation and two gestations. This condition was initially diagnosed using the karyotype technique and finally confirmed with the specialized Fluorescence in-situ hybridization technique. Finally, the quantitative fluorescent polymerase chain reaction technique was used for more certainty. Usually, the male genotype cannot be fertile; since this case is unique, various reasons are involved in its occurrence. One of these cases can be a bone marrow or blood transplant, but according to the study, the possible development of this disorder can be caused by the fetal period.
 Conclusion: As the prior clinical history of the case report revealed that her twin brother died during her mother’s expectancies, it can be assumed that the blood supply between the two fetuses has been shared unevenly or that feto-fetal transfusion has occurred. This incident has caused her to become a chimera with XY karyotype.

The Genetic Approach to Stillbirth: A Systematic Review

ABSTRACT Stillbirth represents a significant burden to individuals and families and has a great variety of causes, but much is still unknown about the underlying etiologies. The overall contribution of genetic abnormalities to stillbirth is not well known; this study was designed to evaluate known genetic causes of unexplained stillbirths, as well as understand the current position and future direction for using genetic and genomic testing in this field. This study was a systematic review of literature published between 1953 and 2020 on genetic testing and stillbirth. Inclusion criteria for studies were human cases, details and specific data on cohorts that included proportion of unexplained fetal deaths, and data on genetic testing and the subsequent results. Exclusion criteria were pregnancy loss before 22 weeks of gestation, focus on known causes of stillbirth, focus on fetal pathology or abnormalities, investigating neonatal death, genetic testing in populations with congenital anomalies or suspected specific syndromes, and those without details on genetic testing. The final review included 9 studies, 1 comparing karyotyping and chromosomal microarray analysis (CMA); 1 comparing karyotyping, CMA, and quantitative fluorescence–polymerase chain reaction; 2 with CMA only; and 5 with exome sequencing data. The studies that examined only CMA found 1 case of copy number variants (CNVs) in 24 cases and 31 CNVs in 96 cases. The largest study included here analyzed 532 stillbirths and compared CMA and karyotyping. Chromosomal microarray analysis showed a higher rate of detection of CNVs, increasing from 5.8% to 8.3% (P = 0.007) when compared with karyotype analysis. One challenge presented by this topic is that some disorders that could contribute to unexplained stillbirth are typically diagnosed when symptoms appear later in life and so are not usually associated with stillbirth or the risk of stillbirth. One example of this could be cardiac rhythm anomalies or other rare genetic variants that lead to stillbirth when appearing in a particular variation. One study included a cohort of unexplained stillbirth after postmortem examination that excluded obvious causes. Genetic analysis showed that several cases carried a variant that was functionally disruptive that may have contributed to stillbirth, but several additional cases that were functionally normal. Genetic variations relating to unexplained stillbirth are a broad and largely unexplored topic. Genetics play a vital role in clinical exploration and treatment of many disorders and in diagnosing causes of fetal mortality and morbidity. This review shows that genetic testing for stillbirth varies greatly according to test availability and guidelines of use, with some tests showing greater diagnostic yield than others. Although these studies incorporated large cohorts of unexplained stillbirths, genetic analysis identified potential causes for only a small percentage. Future research is needed to explain potential genetic causes for stillbirth.

Identification and Validation of Synapse-related Hub Genes after Spinal Cord Injury by Bioinformatics Analysis.

Spinal cord injury (SCI) is a neurological disease with high morbidity and mortality. Previous studies have shown that abnormally expressed synapse-related genes are closely related to the occurrence and development of SCI. However, little is known about the interaction of these aberrantly expressed genes and the molecular mechanisms that play a role in the injury response. Therefore, deeply exploring the correlation between synapse-related genes and functional recovery after spinal cord injury and the molecular regulation mechanism is of great significance. First, we selected the function GSE45006 dataset to construct three clinically meaningful gene modules by hierarchical clustering analysis in 4 normal samples and 20 SCI samples. Subsequently, we performed functional and pathway enrichment analyses of key modules. The results showed that related module genes were significantly enriched in synaptic structures and functions, such as the regulation of synaptic membranes and membrane potential. A protein-protein interaction network (PPI) was constructed to identify 10 hub genes of SCI, and the results showed that Snap25, Cplx1, Stxbp1, Syt1, Rims1, Rab3a, Syn2, Syn1, Cask, Lin7b were most associated with SCI. Finally, these hub genes were further verified by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR) in the spinal cord tissues of the blank group and SCI rats, and it was found that the expression of these hub genes was significantly decreased in the spinal cord injury compared with the blank group (P ≤ 0.05). These results suggest that the structure and function of synapses play an important role after spinal cord injury. Our study helps to understand the underlying pathogenesis of SCI patients further and identify new targets for SCI treatment.

Long noncoding RNA GPRC5D-AS1 in renal cell carcinoma: a molecular mechanism study.

Clear cell renal cell carcinoma (RCC) is the most common subtype of RCC. Although targeted therapy can provide superior treatment outcomes, it is prone to drug resistance, and individual responses to immunotherapy vary greatly. Therefore, finding new diagnostic and therapeutic targets for RCC is of considerable importance. Long noncoding RNA (lncRNA) GPRC5D-AS1 can serve as a biomarker in clinical applications and the prognosis of lung squamous cell carcinoma. However, the specific mechanism of action of lncRNA GPRC5D-AS1 in RCC has not yet been clarified. Therefore, this paper explores the expression of lncRNA GPRC5D-AS1 in the renal cancer cell line 786-0, and conducts a preliminary study of its molecular mechanism. Selecting nude mice for tumor experiments is because of the high genomic and physiological similarity between mice and humans. Conducting tumor research on mice allows for better control of experimental conditions, aiding researchers in more accurately observing and analysing tumor characteristics and responses. Small interfering RNA (siRNA) and plasmid cloning DNA (pcDNA) 3.1 were used to transfect renal cancer cell line 786-0 to silence and overexpress the lncRNA GPRC5D-AS1 gene. Quantitative real-time fluorescence polymerase chain reaction was used to detect the difference in lncRNA GPRC5D-AS1 expression in blank control group, negative control group, siGPRC5D-AS1 group and oeGPRC5D-AS1 group. The effects of silence and overexpression of lncRNA GPRC5D-AS11 on the proliferation of 786-0 cells were detected in cell colony formation experiments; the changes in the migration and invasion of 786-0 cells were detected via cell scratch assay and transwell assay, respectively; the differences in tumor growth between groups were determined via tumorigenesis experiments in nude mice; and the expression of proliferation-related protein [β-catenin, Ki67 and proliferating cell nuclear antigen (PCNA)] and invasion-related protein (N-cadherin and E-cadherin) were detected via Western blotting. Compared with blank control group and negative control group, the siGPRC5D-AS1 group showed a significant decrease in the relative expression of lncRNA GPRC5D-AS1 (P<0.05), a significant increase in the number of proliferating cells and migrating cells (P<0.05), a significant increase in the tumor volume of nude mice (P<0.05), a significant increase in β-catenin, Ki67, PCNA and N-cadherin protein expression (P<0.05), and a significant decrease in E-cadherin protein expression (P<0.05); conversely, these results were opposite for the eGPRC5D-AS1 group. Silencing the expression of lncRNA GPRC5D-AS1 can enhance the proliferation, invasion, and migration ability of renal cancer cell line 786-0, which can be weakened by the overexpression of lncRNA GPRC5D-AS1.

Clinical value assessment for serum hsa_tsr013526 in the diagnosis of gastric carcinoma.

Gastric carcinoma (GC) is a malignant tumor that is detrimental to human health. Transfer RNA-derived small RNAs are a newly identified class of noncoding small RNAs with specific biological functions that are aberrantly expressed in cancer. The aim of this study was to investigate the potential of hsa_tsr013526 as a biomarker for GC. Quantitative real-time fluorescence polymerase chain reaction was used to detect the expression level of hsa_tsr013526. The molecular characteristics of hsa_tsr013526 were verified by agarose gel electrophoresis, Sanger sequencing, and separation of nuclear and cytoplasmic RNA fractions. By testing the receiver operating characteristic (ROC) curves, the diagnostic efficiency of GC using hsa_tsr013526 was determined. Finally, we predicted the downstream of hsa_tsr013526 using functional assays and bioinformatics analysis. Serum expression of hsa_tsr013526 was higher in GC patients than in healthy donors. Serum expression showed differential changes in GC patients, gastritis patients, and healthy donors. Chi-squared tests showed that high expression of hsa_tsr013526 was significantly correlated with T stage, lymphatic metastasis, and tumor node metastasis stage. ROC curve analysis indicated that GC patients could be discriminated from healthy donors or gastritis patients based on their serum levels of hsa_tsr013526. Furthermore, hsa_tsr013526 expression was significantly reduced in postoperative GC patients (p = .0016). High expression of hsa_tsr013526 promotes gastric cancer cell proliferation, invasion, and migration. Serum hsa_tsr013526 was stable and specific, and could be used for dynamic monitoring of GC patients. Therefore, hsa_tsr013526 may be a new biomarker for the diagnosis and postoperative monitoring of GC patients.

Boron homeostasis affects Longan yield: a study of NIP and BOR boron transporter of two cultivars

BackgroundEssential micronutrient Boron (B) plays crucial roles in plant survival and reproduction but becomes toxic in higher quantities. Although plant cells have different B transport systems, B homeostasis is mainly maintained by two transporter protein families: B exporters (BOR) and nodulin-26-like intrinsic proteins (NIP). Their diversity and differential expression are responsible for varied B tolerance among plant varieties and species. Longan is a highly admired subtropical fruit with a rising market in China and beyond. In the present study, we cultured Shixia (SX) and Yiduo (YD), two differently characterized Longan cultivars, with foliar B spray. We analyzed their leaf physiology, fruit setting, B content, and boron transporter gene expression of various tissue samples. We also traced some of these genes' subcellular localization and overexpression effects.ResultsYD and SX foliage share similar microstructures, except the mesophyll cell wall thickness is double in YD. The B spray differently influenced their cellular constituents and growth regulators. Gene expression analysis showed reduced BOR genes expression and NIP genes differential spatiotemporal expression. Using green fluorescent protein, two high-expressing NIPs, NIP1 and NIP19, were found to translocate in the transformed tobacco leaves' cell membrane. NIPs transformation of SX pollen was confirmed using magnetic beads and quantified using a fluorescence microscope and polymerase chain reaction. An increased seed-setting rate was observed when YD was pollinated using these pollens. Between the DlNIP1 and DlNIP19 transformed SX pollen, the former germinated better with increasing B concentrations and, compared to naturally pollinated plants, had a better seed-setting rate in YD♀ × SX♂.ConclusionSX and YD Longan have different cell wall structures and react differently to foliar B spray, indicating distinct B tolerance and management. Two B transporter NIP genes were traced to localize in the plasma membrane. However, under high B concentrations, their differential expression resulted in differences in Jasmonic acid content, leading to differences in germination rate. Pollination of YD using these NIPs transformed SX pollen also showed NIP1 overexpression might overcome the unilateral cross incompatibility between YD♀ × SX♂ and can be used to increase Longan production.

Correlation Between DCAMKL-1 Protein Expression and K-ras Gene Mutation in Colorectal Cancer.

To investigate the correlation between doublecortin and CaM kinase-like-1 (DCAMKL-1) protein expression, K-ras gene mutation, and their impact on patient prognosis in colorectal cancer (CRC). Immunohistochemistry was used to detect the expression of DCAMKL-1 protein in 60 cases of colorectal adenoma, 82 cases of CRC (including 65 cases of lymph node metastasis) and paraffin-embedded paracancerous intestinal mucosal tissue. K-ras gene mutations in primary CRC lesions were detected using an amplification-refractory mutation system and fluorescent polymerase chain reaction. The relationship between DCAMKL-1 protein expression and K-ras gene mutations with the clinicopathological characteristics of patients with CRC was analyzed. Univariate Kaplan‒Meier survival analysis and multivariate Cox regression analysis were performed using follow-up data. The mutation rate of the K-ras gene in 82 cases of CRC was 48.8% (40/82). The positivity rate for the presence of DCAMKL-1 protein in CRC was 70.7% (58/82), significantly higher than that for colorectal adenomas (53.3%; 32/60) and paracancerous intestinal mucosa (0%; 0/82) (P<0.05). The positive expression rate for the presence of DCAMKL-1 protein in 65 patients with lymph node metastasis was higher in the primary lesions (69.2%; 45/65) than in the lymph node metastases (52.3%; 34/65) (χ2=12.087, P=0.001). The K-ras gene mutation status was positively correlated with DCAMKL-1 protein expression (r=0.252, P=0.022). In this study, a potential positive correlation between K-ras gene mutation and DCAMKL-1 protein expression was identified in CRC tissues. The assessment of K-ras gene mutation status and DCAMKL-1 protein expression holds promise for augmenting early diagnosis and prognosis evaluation in CRC. This approach may improve the overall prognosis and survival outcomes for CRC patients.

Monitoring studies of the occurrence of fire blight pathogen in Kazakhstan and identification of antagonistic microorganisms suppressing its development.

The paper presents data on phytosanitary monitoring of garden cenoses for fire blight in the Turkestan, Zhambyl, and Almaty regions of Kazakhstan. The purpose of this study is to assess the phytosanitary situation in various regions of Kazakhstan, determine the extent of fire blight spread, and isolate and identify the fire blight pathogen. During the study, methods such as hypersensitivity, pathogenicity, and fluorescent simplification-based specific hybridization polymerase chain reaction (FLASH-PCR) were used. It was found that in all the surveyed areas, disease foci were identified. For the first time, the fire blight pathogen was detected on fruit crops such as plum, peach, cherry plum, and quince, as well as on wild apricots. 274 plant samples were collected from which microorganisms were isolated. Isolates related to the fire blight pathogen Erwinia amylovora were identified by methods of hypersensitivity, pathogenicity, and FLASH-PCR diagnostics. Of the 156 isolates of microorganisms isolated from apple tree plant samples, 21 inhibited the in vitro growth of E. amylovora to varying degrees. Isolates 16.2 and 19.2 with maximum antagonistic activity were selected, where the pathogen growth inhibition zones were 52.2 ± 2.58 mm and 45.6 ± 0.55 mm, respectively. Based on the obtained sequence of nucleotides of the 16SpRNA gene site, it was found that the selected isolates with high antagonistic activity belonged to the Pseudomonas genus. In the future, based on these isolates, a new biological product for fire blight control can be created and adapted to the natural and climatic conditions of Kazakhstan.

Incremental yield of whole-genome sequencing over chromosomal microarray analysis and exome sequencing for congenital anomalies in prenatal period and infancy: systematic review and meta-analysis.

First, to determine the incremental yield of whole-genome sequencing (WGS) over quantitative fluorescence polymerase chain reaction (QF-PCR)/chromosomal microarray analysis (CMA) with and without exome sequencing (ES) in fetuses, neonates and infants with a congenital anomaly that was or could have been detected on prenatal ultrasound. Second, to evaluate the turnaround time (TAT) and quantity of DNA required for testing using these pathways. This review was registered prospectively in December 2022. Ovid MEDLINE, EMBASE, MEDLINE (Web of Science), The Cochrane Library and ClinicalTrials.gov databases were searched electronically (January 2010 to December 2022). Inclusion criteria were cohort studies including three or more fetuses, neonates or infants with (i) one or more congenital anomalies; (ii) an anomaly which was or would have been detectable on prenatal ultrasound; and (iii) negative QF-PCR and CMA. In instances in which the CMA result was unavailable, all cases of causative pathogenic copy number variants > 50 kb were excluded, as these would have been detectable on standard prenatal CMA. Pooled incremental yield was determined using a random-effects model and heterogeneity was assessed using Higgins' I2 test. Subanalyses were performed based on pre- or postnatal cohorts, cases with multisystem anomalies and those meeting the NHS England prenatal ES inclusion criteria. A total of 18 studies incorporating 902 eligible cases were included, of which eight (44.4%) studies focused on prenatal cohorts, incorporating 755 cases, and the remaining studies focused on fetuses undergoing postmortem testing or neonates/infants with congenital structural anomalies, constituting the postnatal cohort. The incremental yield of WGS over QF-PCR/CMA was 26% (95% CI, 18-36%) (I2 = 86%), 16% (95% CI, 9-24%) (I2 = 85%) and 39% (95% CI, 27-51%) (I2 = 53%) for all, prenatal and postnatal cases, respectively. The incremental yield increased in cases in which sequencing was performed in line with the NHS England prenatal ES criteria (32% (95% CI, 22-42%); I2 = 70%) and in those with multisystem anomalies (30% (95% CI, 19-43%); I2 = 65%). The incremental yield of WGS for variants of uncertain significance (VUS) was 18% (95% CI, 7-33%) (I2 = 74%). The incremental yield of WGS over QF-PCR/CMA and ES was 1% (95% CI, 0-4%) (I2 = 47%). The pooled median TAT of WGS was 18 (range, 1-912) days, and the quantity of DNA required was 100 ± 0 ng for WGS and 350 ± 50 ng for QF-PCR/CMA and ES (P = 0.03). While WGS in cases with congenital anomaly holds great promise, its incremental yield over ES is yet to be demonstrated. However, the laboratory pathway for WGS requires less DNA with a potentially faster TAT compared with sequential QF-PCR/CMA and ES. There was a relatively high rate of VUS using WGS. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Rapid diagnosis of maternal origin of de novo fetal Robertsonian translocation down syndrome of 46,XY,der(13;21)(q10;q10),+21 by quantitative fluorescent polymerase chain reaction in a pregnancy associated with increased nuchal translucency and an abnormal result of first-trimester maternal serum screening

Rapid diagnosis of maternal origin of de novo fetal Robertsonian translocation down syndrome of 46,XY,der(13;21)(q10;q10),+21 by quantitative fluorescent polymerase chain reaction in a pregnancy associated with increased nuchal translucency and an abnormal result of first-trimester maternal serum screening

Human cytomegalovirus glycoprotein B genotypic distributions and viral load in symptomatic infants.

HCMV infection is widespread in humans. This retrospective study aimed to explore the relationship between human cytomegalovirus (HCMV) glycoprotein B (gB) genotype distribution, viral load, and the demographic and clinical features of symptomatic infants. The detection rate of HCMV in blood and urine samples was also compared. Retrospective data from 265 infants who underwent urine HCMV DNA testing were analyzed. The viral load and gB genotype were detected in 91 HCMV positive infants by quantitative fluorescence polymerase chain reaction (PCR) and DNA sequencing, respectively. The positive rate of HCMV infection was 46.04% (122/265) in all infants, and increased rapidly with age. Among the 91 infants investigated, liver function abnormality was the most common diagnosis (34/91, 37.36%), followed by pneumonia (21/91, 23.07%). Sequence analysis of gB yielded two genetic subtypes: the most prevalent gB3 (47/91, 51.65%), followed by gB1 (44/91, 48.35%). The gB3 HCMV infection was more prevalent in infants aged 0-2 months than in infants aged 3-12 months (χ2 = 4.38, p = 0.0364). The data showed that ALT and AST levels were significantly higher in the anti-HCMV IgM+IgG- group than in the anti-HCMV IgM+IgG+ and IgM-IgG+ groups. In addition, this study showed that the detection rate of HCMV DNA in the blood was significantly lower than that in the urine (χ2 = 6.7131, p = 0.0096). This study presents the HCMV infection status of infants and its relationship with their demographic characteristics and clinical manifestations. In addition, this study suggests that urinary PCR is the most appropriate assay for detecting HCMV infections.

Limited additional value of karyotyping cultured amniotic fluid cell colonies in addition to microarray on uncultured cells for confirmation of abnormal non-invasive prenatal testing results.

Non-invasive prenatal testing (NIPT) allows the detection of placental chromosome aberrations. To verify whether the fetus also has the chromosome aberration, diagnostic follow-up testing is required. The aim of this retrospective study was to assess the added value of analyzing amniotic fluid (AF) cell cultures in addition to uncultured AF cells for the detection of fetal mosaicism. NIPT was performed as part of the Dutch TRIDENT study. Cytogenetic studies in uncultured AF were performed using single nucleotide polymorphism (SNP)-array. Cultured AF cell colonies (in situ method) were investigated with fluorescent in situ hybridization and/or karyotyping. Clinical outcome data were collected in cases with discordant results. Between April 2014 and December 2021, 368 amniocenteses were performed after a chromosomal aberration was detected with NIPT. Excluding 134 cases of common aneuploidies (confirmed by quantitative fluorescence polymerase chain reaction), 29 cases with investigation of uncultured cells only and 1 case without informed consent, 204 cases were eligible for this study. In 196 (96%) cases, the results in uncultured and cultured cells were concordant normal, abnormal or mosaic. Five cases (2%) showed mosaicism in cultured AF cells, whereas uncultured AF cells were normal. Two (1%) of these, one mosaic trisomy 13 and one mosaic trisomy 16, were considered true fetal mosaics. The added value of investigating AF cell cultures in addition to uncultured cells is limited to two of 204 (1%) cases in which true fetal mosaicsm would otherwise be missed. The clinical relevance of one (trisomy 13) remained unknown and the other case also showed ultrasound anomalies, which determined pregnancy management. This seems to justify limiting prenatal cytogenetic confirmatory testing to SNP arrays on uncultured AF cells, considerably shortening the reporting time.

The establishment of a multiplex fluorescent polymerase chain reaction coupled with capillary electrophoresis analysis technology enables the simultaneous detection of 16 genotypes of human papillomavirus.

The detection and accurate genotyping of human papillomavirus (HPV) infection is critical for preventing and effectively treating cervical cancer. A multiplex fluorescent polymerase chain reaction (PCR) coupled with a capillary electrophoresis method was developed for the simultaneous detection of the 16 most prevalent HPV genotypes. Twenty-five pairs of primers were ultimately selected to ensure that both E and L regions of nine HPV genotypes, as well as the Eregions of seven HPV genotypes could be accurately amplified. This method enables the simultaneous detection and differentiation of 16 HPV genotypes in a single closed-tube reaction, accurately distinguishing products with molecular weight differences >1 bp through capillary electrophoresis. This method demonstrated exceptional accuracy, specificity, and repeatability with a detection limit of 10 copies/μL for all 16 HPV genotypes. Furthermore, 152 cervical swab specimens were obtained to compare the disparities between this approach and Cobas 4800 HPV detection method. The concordance rate and κ value were 90.1% and 0.802, respectively, indicating a high level of agreement. The established detection method was successfully applied to cervical swab specimens for determining HPV genotypes across all levels of cervical lesions, HPV52, 56, 16, and 59 were found to be most prevalent with infection rates of 10.8%, 9.1%, 6.5%, and 6.2%, respectively. This study has successfully established a detection method capable of simultaneously identifying 16 HPV genotypes. This approach can be further applied to HPV vaccine research and surveillance, with the potential for broad applications.

Genetic analysis of chorionic villus tissues in early missed abortions

Chromosomal abnormalities are the most common etiology of early spontaneous miscarriage. However, traditional karyotyping of chorionic villus samples (CVSs) is limited by cell culture and its low resolution. The objective of our study was to investigate the efficiency of molecular karyotyping technology for genetic diagnosis of early missed abortion tissues. Chromosome analysis of 1191 abortion CVSs in early pregnancy was conducted from August 2016 to June 2021; 463 cases were conducted via copy-number variations sequencing (CNV-seq)/quantitative fluorescent-polymerase chain reaction (QF-PCR) and 728 cases were conducted using SNP array. Clinically significant CNVs of CVSs were identified to clarify the cause of miscarriage and to guide the couples’ subsequent pregnancies. Among these, 31 cases with significant maternal cell contamination were removed from the study. Among the remaining 1160 samples, 751 cases (64.7%) with genetic abnormalities were identified, of which, 531 (45.8%) were single aneuploidies, 31 (2.7%) were multiple aneuploidies, 50 (4.3%) were polyploidies, 54 (4.7%) were partial aneuploidies, 77 (6.6%) had submicroscopic CNVs (including 25 with clinically significant CNVs and 52 had variants of uncertain significance), and 8 cases (0.7%) were uniparental disomies. Our study suggests that both SNP array and CNV-seq/QF-PCR are reliable, robust, and high-resolution technologies for genetic diagnosis of miscarriage.

Modified Danzhi Xiaoyao Powder (MDXP) improves the corneal damage in dry eye disease (DED) mice through phagocytosis

Ethnopharmacological relevanceModified Danzhi Xiaoyao Powder (MDXP) is a traditional Chinese medicine formula remedy for treating Dry Eye Disease (DED). It showed the function of dispersing stagnated liver Qi for relieving Qi stagnation and clearing heat, which can be effective in treating conditions such as Dry Eye Disease (DED) and irregular menstruation due to liver depression and fire transformation. Aim of the studyThis study investigated the mechanism of the effect of MDXP in mice with DED. Materials and methodsA DED model was induced in mice using chronic painful stimulation (tail clamping) in combination with Benzalkonium Chloride Solution drops administered in a dry box for 28 days. After modeling, the MDXP groups were given Chinese medicine with different dosages by gavage for 14 days. The following parameters were recorded in each group: body mass, anal temperature, tear secretion, tear film rupture time, and corneal fluorescein staining. Behavioral changes were evaluated by elevating cross-maze and open-field experiments. The levels of inflammatory factors serum tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), fcγR-mediated phagocytosis pathway cell division control protein 42 homolog (CDC42), actin-related protein 2/3 complex subunit 2 (ARPC2), and actin-related protein 3 (ACTR3) were measured by using Enzyme-linked immunoassay (ELISA), immunohistochemical staining, and real-time fluorescent qualitative polymerase chain reaction (RT-qPCR). ResultsMDXP increased body mass and lowered body temperature, prolonged tear film break-up time, promoted tear secretion, repaired corneal damage, decreased horizontal and vertical scores, elevated percentage of open arm times and boom opening time percentage, and reduced the expression levels of inflammatory factors of TNF-α, IL-1β and pathway-related proteins CDC42, ARPC2, and ACTR3 in mice. MDXP also reduced the expression levels of inflammatory factors of TNF-α and IL-1β in human corneal endothelial cells (HCECs), mouse mononuclear macrophage cells (RAW264.7), and human myeloid leukemia mononuclear cells (THP-1). ConclusionsMDXP can relieve tension and anxiety, inhibit apoptosis, reduce phagocytosis, reduce the expression of pro-inflammatory factors, repair corneal damage, and improve the symptoms in DED mice. The mechanism of action may be through the fcγR-mediated phagocytosis pathway.

Prenatal Screening Using QF-PCR and Karyotyping with an Evaluation of Short Tandem Repeats Markers in the Iranian Population

Background: Quantitative fluorescent Polymerase Chain Reaction (QF-PCR) has been widely used by laboratories as a rapid, low-cost, and convenient test compared to conventional karyotyping for detecting the most common aneuploidies for prenatal diagnosis. Although the latter has been considered the gold standard for detection, the debate to use QF-PCR or both methods together continues.&#x0D; Methods: We screened the results of QF-PCR and karyotyping to compare their detection rate for the most common aneuploidies. In addition, we aimed to investigate the most informative markers in the Iranian population for aneuploidies.&#x0D; Results: We screened 741 pregnant women’s amniotic fluid samples with nuchal translucency (NT) ≥ 2.5 for two years, during which QF-PCR and karyotyping were performed to compare the results. Also, we did a statistical assessment of samples for heterozygosity of 25 short tandem repeats (STR) markers in the Iranian population, which can be applied to find the most informative markers based on the population for each chromosome analyzed in the QF-PCR test.&#x0D; Conclusion: QF-PCR could be used as a stand-alone test to reduce the workload and time-consuming of karyotyping, but using both of them could lead us to the most reliable results.

Epidemiological and viral studies of rabies in Bali, Indonesia.

Rabies has been endemic in Bali since 2009, and cases has recently increased. Unfortunately, there is a lack of available vaccines, which hinders the eradication program. This study aimed to investigate the epidemiological and virological aspects of rabies infection in Bali. A total of 24 brain samples were collected from rabid dogs in all districts of Bali. The samples were tested using the direct fluorescent antibody (DFA) test and polymerase chain reaction (PCR) to confirm the presence of rabies virus in the samples. Samples with the highest virus content were propagated in vivo and then inoculated into BALB/c mice. The brains of dead mice were used to prepare an inoculate cultured in murine neuroblastoma cells. Supernatant-positive viruses representing each district were then reinoculated into eight groups of five BALB/c mice. A brain sample from each dead mouse was tested using DFA and PCR and detected under a fluorescence microscope. All rabies virus-positive samples collected from rabid dogs in all districts of Bali were positive. Rabies virus was detected by DFA test and PCR and was consistently confirmed in the in vivo and in vitro studies. BALB/c mice inoculated with the highest viral dilution (105 cells/mL) of culture supernatant showed typical signs of rabies, indicating that the virus could be properly investigated. This study demonstrated a wide epidemiological distribution of rabies in Bali. The obtained virus can be adapted for in vitro and in vivo studies and can be used to develop a homologous vaccine.