OBJECTIVES: To compare the effects of cell lysis method and magnetic beads method in forensic DNA identification and to explore these two methods in forensic DNA identification. METHODS: The genome DNA of THP-1 cells in different quantities was extracted by the cell lysis method and magnetic beads method, and the DNA content was quantified by real-time quantitative PCR. The cell lysis method and magnetic beads method were used to type the STR of human blood with different dilution ratios. RESULTS: When the numbers of THP-1 cell were 100, 400 and 800, the DNA content extracted by cell lysis method were (1.219±0.334), (5.081±0.335), (9.332±0.318) ng, respectively; and the DNA content extracted by magnetic beads method were (1.020±0.281), (3.634±0.482), (7.896±0.759) ng, respectively. When the numbers of THP-1 cells were 400 and 800, the DNA content extracted by the cell lysis method was higher than that by the magnetic beads method. The sensitivity of cell lysis method and magnetic beads method was similar in STR typing of human blood at different dilution ratios. Complete STR typing could be obtained at 100, 300 and 500-fold dilutions of blood samples, but could not be detected at 700-fold dilution. STR typing of undiluted human blood could not be detected by cell lysis method. CONCLUSIONS The cell lysis method is easy to operate and can retain template DNA to the maximum extend. It is expected to be suitable for trace blood evidence tests.
Humans ; Forensic Medicine ; DNA/genetics* ; Real-Time Polymerase Chain Reaction ; Magnetic Phenomena ; DNA Fingerprinting/methods* ; Microsatellite Repeats

Mycoplasma species (spp.) are bacteria that are difficult to detect. Currently, the polymerase chain reaction (PCR) is considered the most effective diagnostic tool to detect these microorganisms in both human and veterinary medicine. There are 13 known species of human Mycoplasma and 15 species of canine Mycoplasma. Owing to the difficulties in identifying the individual species of Mycoplasma, there is a lack of information regarding which species are saprophytic and which are pathogenic. The prevalence of the individual species is also unknown. In addition, in both humans and dogs, the results of some studies on the impact of Mycoplasma are conflicting. The presence of Mycoplasma spp. on the epithelium of reproductive tract is often associated with infertility, although they are also detected in healthy individuals. The occurrence of Mycoplasma spp. is more common in dogs (even 89%) than in humans (1.3%-4%). This is probably because the pH of a dog's genital is more conducive to the growth of Mycoplasma spp. than that of humans. Phylogenetically, human and canine Mycoplasma are related, and majority of them belong to the same taxonomic group. Furthermore, 40% of canine Mycoplasma spp. are placed in common clusters with those of human. This suggests that species from the same cluster can play a similar role in the canine and human reproductive tracts. This review summarizes the current state of knowledge about the impact of Mycoplasma on canine and human male fertility as well as the prospects of further development in this field.
Humans ; Dogs ; Male ; Animals ; Mycoplasma/genetics* ; Infertility ; Semen Analysis ; Polymerase Chain Reaction/methods* ; Prevalence ; Semen/chemistry*

OBJECTIVE: To confirm the HLA genotypes of the samples including 4 cases of magnetic bead probe HLA genotyping result pattern abnormality and 3 cases of ambiguous result detected by PCR sequence-specific oligonudeotide probe (SSOP) method. METHODS: All samples derived from HLA high-resolution typing laboratory were detected by PCR-SSOP. A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality and 3 samples of ambiguous result were further confirmed by PCR sequence-based typing (SBT) technology and next-generation sequencing (NGS) technology. RESULTS: A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality were detected by PCR-SSOP method. The results of SBT and NGS showed that the HLA-A genotype of sample 1 did not match any known genotypes. NGS analysis revealed that the novel allele was different from the closest matching allele A*31:01:02:01at position 154 with G>A in exon 2, which resulting in one amino acid substitution at codon 28 from Valine to Methionine (p.Val28Met). The HLA-C genotype of sample 2 was C*03:119, 06:02, sample 3 was C*03:03, 07:137, and sample 4 was B*55:02, 55:12. A total of 3 samples with ambiguous result were initially detected by PCR-SSOP method. The re-examination results of SBT and NGS showed that the HLA-B genotype of sample 5 was B*15:58, 38:02, sample 6 was DRB1*04:05, 14:101, and sample 7 was DQB1*03:34, 05:02. Among them, alleles C*03:119, C*07:137 and DRB1*14:101 were not included in the Common and Well-documented Alleles (CWD) v2.4 of the Chinese Hematopoietic Stem Cell Donor Database. CONCLUSION The abnormal pattern of HLA genotyping results of magnetic probe by PCR-SSOP method suggests that it may be a rare allele or a novel allele, which needs to be verified by sequencing.
Humans ; Alleles ; Polymerase Chain Reaction ; Genotype ; High-Throughput Nucleotide Sequencing ; Histocompatibility Testing/methods* ; Technology

Objective: To establish a rapid and specific quantitative real-time PCR (qPCR) method for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA (SgN) in patients with COVID-19 or environmental samples. Methods: The qPCR assay was established by designing specific primers and TaqMan probe based on the SARS-CoV-2 genomic sequence in Global Initiative of Sharing All Influenza Data (GISAID) database. The reaction conditions were optimized by using different annealing temperature, different primers and probe concentrations and the standard curve was established. Further, the specificity, sensitivity and repeatability were also assessed. The established SgN and genomic RNA (gRNA) qPCR assays were both applied to detect 21 environmental samples and 351 clinical samples containing 48 recovered patients. In the specimens with both positive gRNA and positive SgN, 25 specimens were inoculated on cells. Results: The primers and probes of SgN had good specificity for SARS-CoV-2. The minimum detection limit of the preliminarily established qPCR detection method for SgN was 1.5×10² copies/ml, with a coefficient of variation less than 1%. The positive rate of gRNA in 372 samples was 97.04% (361/372). The positive rates of SgN in positive environmental samples and positive clinical samples were 36.84% (7/19) and 49.42% (169/342), respectively. The positive rate and copy number of SgN in Wild strain were lower than those of SARS-CoV-2 Delta strain. Among the 25 SgN positive samples, 12 samples within 5 days of sampling time were all isolated with virus; 13 samples sampled for more than 12 days had no cytopathic effect. Conclusion: A qPCR method for the detection of SARS-CoV-2 SgN has been successfully established. The sensitivity, specificity and repeatability of this method are good.
Humans ; SARS-CoV-2/genetics* ; COVID-19/diagnosis* ; Subgenomic RNA ; Real-Time Polymerase Chain Reaction/methods* ; RNA, Viral/genetics* ; Sensitivity and Specificity ; Nucleocapsid/chemistry* ; COVID-19 Testing

Gelsemium elegans is a traditional Chinese herb of medicinal importance, with indole terpene alkaloids as its main active components. To study the expression of the most suitable housekeeping reference genes in G. elegans, the root bark, stem segments, leaves and inflorescences of four different parts of G. elegans were used as materials in this study. The expression stability of 10 candidate housekeeping reference genes (18S, GAPDH, Actin, TUA, TUB, SAND, EF-1α, UBC, UBQ, and cdc25) was assessed through real-time fluorescence quantitative PCR, GeNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that EF-1α was stably expressed in all four parts of G. elegans and was the most suitable housekeeping gene. Based on the coexpression pattern of genome, full-length transcriptome and metabolome, the key candidate targets of 18 related genes (AS, AnPRT, PRAI, IGPS, TSA, TSB, TDC, GES, G8H, 8-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, SLS, STR, and SGD) involved in the Gelsemium alkaloid biosynthesis were obtained. The expression of 18 related enzyme genes were analyzed by qRT-PCR using the housekeeping gene EF-1α as a reference. The results showed that these genes' expression and gelsenicine content trends were correlated and were likely to be involved in the biosynthesis of the Gelsemium alkaloid, gelsenicine.
Genes, Essential ; Gelsemium/genetics* ; Peptide Elongation Factor 1/genetics* ; Transcriptome ; Gene Expression Profiling/methods* ; Alkaloids ; Real-Time Polymerase Chain Reaction/methods* ; Reference Standards

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.
Real-Time Polymerase Chain Reaction/methods* ; Paeonia/genetics* ; Actins/genetics* ; Reproducibility of Results ; Transcriptome ; Glyceraldehyde-3-Phosphate Dehydrogenases/genetics* ; Reference Standards ; Gene Expression Profiling/methods*

Cytosine methylation is one of the major types of DNA epigenetic modifications and plays an important role in maintaining normal cell function and regulating gene expression. Bisulfite sequencing PCR (BSP) based cloning and sequencing is a general method for detecting DNA methylation at specific sites, which can clarify the methylation status of each CpG site in the target fragment. However, this method requires large amounts of single-clonal sequencing, which is complicated to operate, time consuming and expensive. Therefore, the development of an accurate, efficient and convenient DNA methylation detection technology is of great significance to improve the efficiency of epigenetic research. Based on the high-throughput mutation detection platform Hi-TOM (high-throughput tracking of mutations) developed by our group, we further established a site-specific DNA methylation high-throughput detection platform Hi-Meth (High-throughput Detection of DNA Methylation). After bisulfite treatment of DNA samples, the specific site-specific DNA methylation analysis results could be obtained through the Hi-Meth platform by performing only one round of PCR amplification. Using the Hi-Meth platform, the DNA methylation status of two promoter regions of rice were detected. The DNA methylation results from Hi-Meth were consistent with the results from BSP-based method. Thus, site-specific DNA methylation analysis results could be obtained accurately and conveniently through the Hi-Meth platform. In conclusion, Hi-Meth provides an important methylation detection platform for specific DNA regions, which has important significance for epigenetic research.
DNA Methylation ; Epigenesis, Genetic ; Epigenomics ; High-Throughput Nucleotide Sequencing/methods* ; Polymerase Chain Reaction ; Sequence Analysis, DNA/methods*

OBJECTIVE: Three cases of rare alleles of HLA-C with zero mismatched PCR-SBT results were analyzed by full-length sequencing to determine the true genotypes. METHODS: Three rare HLA-C alleles with zero mismatched PCR-SBT results were screened from clinical transplant matching samples, and the full-length sequence was detected by next-generation sequencing technology. RESULTS: The results of PCR-SBT typing of 3 samples were: HLA-C*03:04, 12:167; HLA-C*07:291, 15:02; HLA-C*01:43, 08:16. Other alleles were not in the CWD table of common and confirmed HLA alleles in China (version 2.3) except common allele HLA-C*03:04, HLA-C*15:02. NGS full-length sequencing revealed that the HLA-C genotypes of the three samples were a combination of common alleles and novel alleles, and the three novel alleles had a base mutation in exons 6, 2, and 4, respectively. The novel allele sequences have been submitted to the Genbank database (MK629722, MK335474, MK641803), which were officially named HLA-C*03:04:74, HLA-C*15:192, HLA-C*08:01:25 by the WHO HLA Nomenclature Committee. The HLA high-resolution typing results of 3 samples were: HLA-C*03:04:74, HLA-C*12:03; HLA-C*07:02, HLA-C*15:192; HLA-C*01:02, HLA-C*08:01:25. CONCLUSION HLA typing results containing rare alleles should be treated cautiously, and the full-length sequence should be verified by NGS or cloning. The laboratory finally confirmed that the 3 cases of PCR-SBT zero mismatch HLA-C genotypes are the combination of common alleles and novel alleles by NGS sequencing, which provides an accurate basis for clinical transplantation matching and enriches the human HLA genetic database.
Alleles ; Genotype ; HLA-C Antigens/genetics* ; High-Throughput Nucleotide Sequencing ; Histocompatibility Testing/methods* ; Humans ; Polymerase Chain Reaction/methods* ; Sequence Analysis, DNA

OBJECTIVE: To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation. METHODS: Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 ^-/-, arhgef10 ^+/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively. RESULTS: WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 ^-/- zebrafish had more severe symptoms than arhgef10 ^+/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis. CONCLUSION Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.
Animals ; Annexin A5 ; Apoptosis ; Blotting, Western ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Cells, Cultured ; Flow Cytometry ; Genotype ; Humans ; In Situ Hybridization ; Larva/physiology* ; Phenotype ; RNA/isolation & purification* ; Real-Time Polymerase Chain Reaction/standards* ; Rho Guanine Nucleotide Exchange Factors/metabolism* ; Sincalide/analysis* ; Spectrophotometry/methods* ; Zebrafish/physiology*

DNA polymerases are widely used in PCR and play important roles in life science research and related fields. Development of high-performance DNA polymerases is of great commercial interest as the current commercial DNA polymerases could not fully satisfy the requirements of scientific research. In this study, we cloned and expressed a family B DNA polymerase (NCBI accession number TEU_RS04875) from Thermococcus eurythermalis A501, characterized its enzymatic property and evaluated its application in PCR. The recombinant Teu-PolB was expressed in E. coli and purified with affinity chromatography and ion-exchange chromatography. The enzymatic properties of Teu-PolB were characterized using fluorescence-labeled oligonucleotides as substrates. The application potential of Teu-PolB in PCR was evaluated using the phage λ genomic DNA as a template. Teu-PolB has DNA polymerase and 3'→5' exonuclease activities, and is highly thermostable with a half-life of 2 h at 98 ℃. The most suitable PCR buffer is consisted of 50 mmol/L Tris-HCl pH 8.0, 2.5 mmol/L MgCl₂, 60 mmol/L KCl, 10 mmol/L (NH₄)₂SO₄, 0.015% Triton X-100 and 0.01% BSA, and the optimal extension temperature is 68 ℃. Under the optimized conditions, a 4 kb target fragment was successfully amplified with an extension rate of 2 kb/min. The yield of the Teu-PolB amplified-DNA was lower than that of Taq DNA polymerase, but its extension rate and fidelity was higher than that of Taq and Pfu DNA polymerases. The biochemical properties of Teu-PolB demonstrate that this enzyme can be used in PCR amplification with high thermostability, good salt tolerance, high extension rate and high fidelity.
DNA-Directed DNA Polymerase/genetics* ; Escherichia coli/genetics* ; Polymerase Chain Reaction/methods* ; Temperature ; Thermococcus/genetics*