This study aims to establish a high-performance size-exclusion chromatography (HPSEC) method for determining the content of the classical swine fever virus (CSFV) E2 protein and screen the optimal stabilizer to enhance the stability of this protein. The optimal detection conditions were determined by optimizing the composition of the mobile phase, and characteristic chromatographic peaks were identified by SDS-PAGE and Western blotting. The specificity, repeatability, precision, linearity, limit of detection (LOD), and limit of quantitation (LOQ) of the method were assessed. The method established was used to determine the content of CSFV E2 protein antigen and vaccine. Differential scanning fluorimetry (DSF) was employed to screen the buffer system, pH, and salt ion concentrations, and sugar, amino acid, and alcohol stabilizers were further screened. The results showed that using a 200 mmol/L phosphate buffer provided the best column efficiency. An antigen-specific chromatographic peak appeared at the retention time of 18 min, which was identified as the CSFV E2 protein by SDS-PAGE and Western blotting. The method exhibited high specificity for detecting the CSFV E2 protein, with no absorbance peak observed in the blank control. The relative standard deviation (RSD) of the peak area for six repeated injections of the CSFV E2 protein was 0.74%, indicating good repeatability of the method. The RSD for repeated detection of two different concentrations of CSFV E2 protein samples by different operators at different time points was less than 2%, suggesting good intermediate precision of the method. The peak area of the CSFV E2 protein was linearly related to its concentration, with the regression equation showing R² of 1.000. The LOD and LOQ of the method were 14.88 μg/mL and 29.75 μg/mL, respectively. Application of the developed method in the detection of three batches of CSFV E2 protein antigen and three batches of vaccine demonstrated results consistent with those from the bicinchoninic acid (BCA) assay, which meant that the method could accurately determine the content of CSFV E2 protein antigen and vaccine. The DSF method identified 50 mmol/L Tris-HCl at pH 8.0 as the optimal buffer, and the addition of sugar and alcohol stabilizers further improved the stability of the CSFV E2 protein. The HPSEC method established in this study is simple, fast, and exhibits good accuracy and repeatability, enabling precise measurement of the CSFV E2 protein content. It is expected to play a crucial role in the quality control of the CSFV E2 vaccine. Furthermore, the strategy for improving the CSFV E2 protein stability, identified through DSF screening, has significant implications for enhancing the stability of the CSFV E2 vaccine.
Classical Swine Fever Virus/chemistry* ; Chromatography, Gel/methods* ; Animals ; Swine ; Viral Envelope Proteins/immunology*

OBJECTIVE: To explore the genetic basis for child with congenital cataract. METHODS: The child was subjected to next-generation sequencing. Candidate variant was verified by Sanger sequencing of his family members. RESULTS: The proband was found to harbor novel heterozygous variants of c.855del and c.872dup of the GJA8 gene, which were inherited from his father and mother, respectively. Neither of these two variants has been reported. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, the c.855del and c.872dup variants were classified as likely pathogenic (PVS1_S+PM2+PP4) and pathogenic (PVS1_S+PM2+PM3+PP4), respectively. CONCLUSION The c.855del and c.872dup variants of the GJA8 gene probably underlay the congenital cataract in this patient.
Child ; Humans ; Cataract/congenital* ; Family ; Heterozygote ; High-Throughput Nucleotide Sequencing ; Mutation ; Pedigree

Objective:To establish the method for detecting lower respiratory infections (LRIs) bacterialpathogens using nanopore sequencing, and evaluate the feasibility of this method.Methods:Bronchoalveolar lavage fluid (BALF) samples from 33 patients with LRIs who visited the Department of Respiratory and Critical Care Medicine of Beijing Hospital from July 2019 to September 2020 were collected.Nanopore 16S amplicon sequencing were performed on these samples. In order to evaluate the clinical value of the nanopore sequencing, χ 2 test was used to analyze the pathogen differences between the detection rate and pathogen types results found with using the nanopore 16S sequencing and the results found with bacterial culture. Results:The process and method of nanopore sequencing used in the detection of the LRIs pathogens were established. The pathogen detection rate of the 16S sequencing was higher than that of the traditional bacterial culture (75.8% [25/33], 45.5% [15/33], χ2=5.140, P<0.05). From the 25 positive samples found with nanopore 16S sequencing, 16 pathogens were detected, including Haemophilus parainfluenzae, Haemophilus influenzae, Streptococcus pneumoniae, Streptomonas maltophilia, Acinetobacter baumannii, and Acinetobacter junii, Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, Enterococcus gallinarum, Corynebacterium striatum, Mycobacterium paraintracellulare, Serratia marcescens, Achromobacter insuavis, Citrobacter murliniae and Mycoplasma pneumoniae. More than 6 pathogens were tested in clinical culture, including Haemophilus parainfluenzae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae and Streptomonas maltophilia (χ2=7.949, P<0.05). 16S sequencing aligned to species level sequences accounted for 80.0 (60.0, 86.0)% of the genus level. The results obtained by using16S sequencing and bacterial culture were consistent in 11 (33.3%) samples. Conclusions:Nanopore 16S amplicon sequencing can quickly identify pathogenic bacteria from BALF in LRIs patients. Nanopore 16S amplicon sequencing has a high detection rate, it can detect more pathogens than traditional bacterial culture, and it can also identify most bacteria to the species level. This technology is a very promising platform with broad application prospects.

OBJECTIVE: To explore the genetic basis for a patient with tuberous sclerosis complex. METHODS: Genomic DNA was extracted from peripheral blood samples from members of his family and 100 unrelated healthy controls. The proband was subjected to next-generation sequencing, and candidate variant was confirmed by multiple ligation-dependent probe amplification (MLPA) and Sanger sequencing. Reverse transcription-PCR (RT-PCR) was carried out to determine the relative mRNA expression in the proband. RESULTS: The patient was found to harbor a c.2355+1G>C splicing variant of the TSC2 gene. Sequencing of cDNA confirmed that 62 bases have been inserted into the 3' end of exon 21, which has caused a frameshift producing a truncated protein. CONCLUSION The novel splicing variant c.2355+1G>C of the TSC2 gene probably underlay the TSC in the proband. Above finding has expanded the variant spectrum of TSC2 and provided a basis for preimplantation genetic testing and/or prenatal diagnosis.
Female ; Humans ; Mutation ; Pregnancy ; RNA Splicing/genetics* ; Tuberous Sclerosis/genetics* ; Tuberous Sclerosis Complex 1 Protein/genetics* ; Tuberous Sclerosis Complex 2 Protein/genetics*

OBJECTIVE: To explore the genetic basis for a pedigree affected with Nance-Horan syndrome. METHODS: Clinical manifestation of the patients was analyzed. Genomic DNA was extracted from peripheral blood samples of the pedigree members and 100 unrelated healthy controls. A panel of genes for congenital cataract was subjected to next-generation sequencing (NGS), and candidate variant was verified by Sanger sequencing and bioinformatic analysis based on guidelines of American College of Medical Genetics and Genomics (ACMG). mRNA expression was determined by reverse transcriptase-PCR (RT-PCR). Linkage analysis based on short tandem repeats was carried out to confirm the consanguinity. RESULTS: A small insertional variant c.766dupC (p.Leu256Profs*21) of the NHS gene was identified in the proband and his affected mother, but not among unaffected members and the 100 healthy controls. The variant was unreported in Human Gene Mutation Database (HGMD) and other databases. Based on the ACMG guideline, the variant is predicted to be pathogenic (PVS1+PM2+PM6+PP4). CONCLUSION The novel variant c.766dupC of the NHS gene probably underlay the X-linked dominant Nance-Horan syndrome in this pedigree.
Cataract/genetics* ; Genetic Diseases, X-Linked ; Humans ; Mutation ; Pedigree ; State Medicine ; Tooth Abnormalities

To investigate whether the laboratory specimens preserved in Beijing Hospital Biobank during a specific period had been contaminated by SARS-Cov-2 through a cross-sectional study, and to establish a retrospective biobank safety screening system. Laboratory specimens were collected from the Department of Respiratory and Critical Care Medicine and the Fever Clinic of Beijing Hospital from November 1, 2019 to January 22, 2020, nucleic acid and serological antibody testing were performed for SARS-CoV-2 in these specimens (including 79 serum, 20 urine, 42 feces and 21 bronchoalveolar lavage fluid specimens). The safety of the stored samples during this period was defined by negative and positive results. Both the nucleic acid test and serological antibody test showed negative for SARS-CoV-2, indicating that these specimens were safely stored in the biobank. High-risk specimens collected in our hospital during the early stage of the COVID-19 outbreak are free of SARS-CoV-2, and a safety screening strategy for the clinical biobank is established to ensure the biosafety of these samples.
Biological Specimen Banks ; COVID-19 ; Cross-Sectional Studies ; Hospitals ; Humans ; Retrospective Studies ; SARS-CoV-2

OBJECTIVE: To explore the genetic basis for a pedigree affected with Alport syndrome. METHODS: Next generation sequencing and Sanger sequencing was carried out to detect potential variant of the COL4A5 gene among members from the pedigree and 100 unrelated healthy controls. RESULTS: A novel missense c.3293G>T (p.Gly1098Val) variant was found in the COL4A5 gene among 6 affected members but not the unaffected members of the pedigree or the 100 healthy controls. According to the American College of Medical Genetics and Genomics standards and guidelines, the c.3293G>T variant was classified as pathogenic (PP1-strong+PM1+PM2+PP3+PP4). CONCLUSION By destructing the Gly-X-Y structure of its protein product, the c.3293G>T variant of the COL4A5 gene probably underlies the Alport syndrome in this pedigree. Above finding has enriched the spectrum of COL4A5 variants.

OBJECTIVE: To analyze variant of IDS gene in a pedigree affected with mucopolysaccharidosis type II (MPS II). METHODS: The proband was subjected to next generation sequencing and Sanger sequencing to identify potential variants. Suspected variant was analyzed by its co-segregation with the disease in the pedigree. Its impact on mRNA splicing was analyzed by using reverse transcription PCR (RT-PCR). RESULTS: A hemizygous IVS1-3T>G variant was found in the IDS gene in the proband. RT-PCR results revealed two abnormal cDNA fragments of 600 bp and 300 bp. The 600 bp fragment had inserted 216 nucleotides at the 3' end of intron 1, while the 300 bp fragment had lost 109 nucleotides at the 5' end of exon 2, which resulted in two truncated proteins comprising 38 and 92 amino acids, respectively, instead of the normal product (550 amino acids). The proband and his mother were respectively hemizygous and heterozygous for the variant. The same variant was not found among 100 normal controls. CONCLUSION The IVS1-3T>G variant of the IDS gene probably underlies the MPS II in this pedigree by causing reduction or elimination of the IDS protein.

OBJECTIVE: To detect potential variants of MECP2 gene in three pedigrees affected with Rett syndrome (RTT). METHODS: All exons and their flanking regions of the MECP2 gene were subjected to Sanger sequencing and multiplex ligation-dependent probe amplification assay. RESULTS: The probands of pedigrees 1 and 2 have respectively carried a c.965C>G and a c.1157_1197del41 variant of the MECP2 gene, while the proband of pedigree 3 carried a heterozygous deletional variant in exon 4 of the MECP2 gene. CONCLUSION Variants of the MECP2 gene probably underlay the RTT in the three pedigrees. Above finding has enriched the spectrum of MECP2 gene variants, and provided a guidance for the patients upon preimplantation genetic testing and prenatal diagnosis.

OBJECTIVETo assess the value of droplet digital PCR (ddPCR) for non-invasive prenatal diagnosis of single gene disease in two families.

METHODSPaternal mutation in cell-free DNA derived from the maternal blood and amniotic fluid DNA was detected by ddPCR. Suspected mutation in the amniotic fluid DNA was verified with Sanger sequencing.

RESULTSThe result of ddPCR and Sanger sequencing indicated that the fetuses have carried pathogenic mutations from the paternal side in both families.

CONCLUSIONDroplet digital PCR can accurately detect paternal mutation carried by the fetus, and it is sensitive and reliable for analyzing trace samples. This method may be applied for the diagnosis of single gene diseases caused by paternal mutation using peripheral blood sample derived from the mother.

Fathers ; Female ; Genetic Diseases, Inborn ; diagnosis ; Humans ; Male ; Maternal Serum Screening Tests ; Mutation ; Polymerase Chain Reaction ; methods ; Prenatal Diagnosis ; methods ; Sequence Analysis, DNA