Tujia ethnic group medicine Xuetong is derived from Kadsura heteroclita, the stem of which has the medicinal value for anti-rheumatoid arthritis, liver protection, anti-tumor, anti-oxidation effects, and has been widely used in Hunan and Guangdong in China. The selection of reliable and stable reference genes is the basis for subsequent molecular research on K. heteroclita. In this study, GAPDH, TUA, Actin, UBQ, EF-1α, 18S-rRNA, CYP, UBC, TUB, H2A, and RPL were selected as candidate reference genes in Kadsura heteroclita. The gene expression levels of the 11 candidate reference genes of K. heteroclita in its 6 different parts(stem-inside of the cambium, stem-outside of the cambium, fruit, flower, root, and leaf) and under different intervention conditions [drought stress, salt stress, and methyl jasmonate(MeJA) treatment] were detected by quantitative real-time polymerase chain reaction(qRT-PCR). The expression stability of the 11 candidate reference genes was comprehensively analyzed and evaluated by geNorm, NormFinder, ΔCT algorithm, and RefFinder software. The results showed that the expression of UBC and RPL was relatively stable in 6 different parts, and UBC and GAPDH genes were relatively stable under different intervention conditions. To verify the reliability of reference genes for K. heteroclita, this study further examined the relative expression levels of KhFPS, KhIDI, KhCAS, KhSQE, KhSQS, KhSQS-2, KhHMGS, KhHMGR, KhMVD, KhMVK, KhDXR, KhDXS, KhPMVK, and KhGGPS in different parts and under different intervention conditions, which might relate to the synthesis of the main component(Xuetongsu) of K. heteroclita. The results showed that with UBC and RPL or UBC and GAPDH as the reference genes, the expression trends of these 14 genes were basically consistent in different parts or under different intervention conditions for K. heteroclita. In conclusion, UBC can be used as a reference gene of K. heteroclita for its different parts and different intervention conditions, which lays a foundation for further research on the biosynthetic pathway of main components in K. heteroclita.
Real-Time Polymerase Chain Reaction/methods* ; Reference Standards ; Gene Expression Regulation, Plant ; Gene Expression Profiling ; Plant Proteins/metabolism* ; Drugs, Chinese Herbal

OBJECTIVE: To establish a genotyping method for Diego, MNS and Kell blood groups based on quantitative real-time PCR (qPCR) technology, and preliminarily apply it to the screening of rare blood groups in blood donors. METHODS: Blood group gene standards containing heterozygous and homozygous alleles were prepared by blood group serological and PCR-SBT methods. Specific amplification primers and hybridization probes were designed, and explore to establish the qPCR method for detecting Diego, MNS, and Kell blood group genotypes. Then the established qPCR method was used to identify blood group genotypes of 186 blood donor samples. RESULTS: A method based on qPCR technology was established to identify Dia/Dib, S/s and K/k blood group antigens. The genotyping results of the gene standard samples were consistent with the serological testing results and genotypes detected by PCR-SBT. qPCR testing of 186 samples identified 11 cases of DI*A/B heterozygosity and 19 cases of GYPB*S/s heterozygosity, and the rest were DI*B/B, GYPB*s/s, KEL*02/02 homozygosity. No rare blood group genotypes of DI*A/A, GYPB*S/S, KEL*01.01/01.01 were found. CONCLUSION The established qPCR method is suitable for genotyping on Diego, MNS and Kell blood group, and it can be used for batch screening of blood donors and the establishment of rare blood group bank.
Humans ; Genotype ; Genotyping Techniques/methods* ; Real-Time Polymerase Chain Reaction/methods* ; Blood Group Antigens/genetics* ; Kell Blood-Group System/genetics* ; Blood Donors ; Blood Grouping and Crossmatching/methods* ; Erythrocytes ; MNSs Blood-Group System/genetics*

From the perspective of performance evaluation, this paper describes briefly the concerns of study on each component module and the entire instrument, clinical items, software, and product testing on droplet digital PCR instruments, including the study methods and quality control requirements. The increase of the research and development efficiency of products and contribute to the promotion of application of digital PCR instruments in clinical laboratories are expected.
Polymerase Chain Reaction/methods* ; Quality Control ; Software ; Humans

OBJECTIVES: To detect prfA and hly toxin genes of Listeria monocytogenes using polymerase chain reaction (PCR) and colloidal gold technology. METHODS: L. monocytogenes DNA was extracted by boiling method. With prfA and hly of L. monocytogenes as the target genes, the 5' ends of upstream and downstream primers of prfA gene were labeled with 6-FAM and biotin, and the 5' ends of upstream and downstream primers of hly gene were labeled with digoxin and biotin, respectively, to establish the toxin gene detection method. Using cloning transformation, sequencing analysis, cloning of positive control products, the detection kid was developed and its specificity, sensitivity, reproducibility and stability were tested, followed by verification with sample testing. RESULTS: The concentration of L. monocytogenes DNA extracted by boiling method was 148.81±0.97 ng/μL, and the A₂₆₀/A₂₈₀ ratio ranged from 1.8 to 2.0. The PCR products showed a 100% homology with the gene sequences in GenBank database after cloning, transformation and sequencing. The colloidal gold strip yielded positive results only for L. monocytogenes samples without cross-reactions with Staphylococcus aureus, Escherichia coli or Bacillus cereus, and its minimum detection limit was 10^-2 ng/μL, demonstrating a 10-fold greater sensitivity of the test than agarose gel electrophoresis. The test also showed good reproducibility of the results when performed by different operators with good stability of the test strips after storage for 6 to 12 months. The test results showed that this kit could accurately and quickly detect L.monocytogenes in the test samples. CONCLUSIONS The detection kit developed in this study can simultaneously detect prfA and hly toxin genes of L. monocytogenes with good specificity, sensitivity, reproducibility and stability for use in food safety inspection.
Listeria monocytogenes/isolation & purification* ; Gold Colloid ; Bacterial Toxins/genetics* ; Polymerase Chain Reaction/methods* ; Hemolysin Proteins/genetics* ; Bacterial Proteins/genetics* ; DNA, Bacterial/genetics* ; Food Microbiology ; Heat-Shock Proteins

Gene synthesis is an enabling technology that supports the development of synthetic biology. The existing approaches for de novo gene synthesis generally have tedious operation, low efficiency, high error rates, and limited product lengths, being difficult to support the huge demand of synthetic biology. The assembly and error correction are the keys in gene synthesis. This study first designed the oligonucleotide sequences by reasonably splitting the virus genome of approximately 10 kb by balancing the parameters of sequence design software ability, PCR amplification ability, and assembly enzyme assembly ability. Then, two-step PCR was performed with high-fidelity polymerase to complete the de novo synthesis of 3.0 kb DNA fragments, and error correction reactions were performed with T7 endonuclease Ⅰ for the products from different stages of PCR. Finally, the virus genome was assembled by 3.0 kb DNA fragments from de novo synthesis and error correction and then sequenced. The experimental results showed that the proposed method successfully produced the DNA fragment of about 10 kb and reduced the probability of large fragment mutations during the assembly process, with the lowest error rate reaching 0.36 errors/kb. In summary, this study developed an efficient de novo method for synthesizing a viral genome of about 10 kb with T7 endonuclease Ⅰ-mediated error correction. This method enabled the synthesis of a 10 kb viral genome in one day and the correct plasmid of the viral genome in five days. This study optimized the de novo gene synthesis process, reduced the error rate, simplified the synthesis and assembly steps, and reduced the cost of viral genome assembly.
Genome, Viral/genetics* ; Polymerase Chain Reaction/methods* ; DNA, Viral/genetics* ; Bacteriophage T7/enzymology* ; Synthetic Biology/methods*

Calcined oyster is a commonly used shellfish traditional Chinese medicine in clinical practice in China. During the processing of oysters, their microscopic characteristics are destroyed, and open-fire calcination can damage the DNA of oysters, making it difficult to identify the primary source. The establishment of a specific polymerase chain reaction(PCR) method for the identification of calcined oysters can provide a guarantee for the safety and clinical efficacy of the medicine and its processed products. With Ostrea gigas as an example, the DNA extraction method of decoction pieces and formula particles of calcined oysters was improved, and high-quality DNA was obtained. Based on the specific single nucleotide polymorphism(SNP) sites of O. gigas and the other two species, the specific identification primers were designed, and the site-specific identification method of formula granules of calcined oyster(O. gigas) was established. The specificity and applicability of the method were investigated. The results showed that when the annealing temperature was 54 ℃, and the cycle was 44 times, the PCR amplified products of calcined oyster(O. gigas) and its formula granules produced a single bright identification band at 102 bp, while the other two species of oysters, O. talienwhanensis Crosse and O. rivularis Gould, had no band. In this study, DNA extraction and PCR identification of animal medicinal materials by calcination were established for the first time, which provided a tool for solving the difficult identification of calcined decoction pieces and ensuring drug safety.
Animals ; Ostrea/classification* ; Polymerase Chain Reaction/methods* ; Polymorphism, Single Nucleotide ; DNA/genetics*

OBJECTIVE: Assessing the accuracy of automated EasyNAT system for rapidly detecting paraffin-embedded tissue for the diagnosis of tuberculosis. METHODS: A retrospective analysis was conducted on 134 patients, comprising 101 with confirmed tuberculosis and 33 without tuberculosis, treated at Beijing Chest Hospital, Capital Medical University, between 2018 and 2022.The clinical diagnostic results served as the standard for assessing the diagnostic performance of the EasyNAT system in comparison to quantitative real-time polymerase chain reaction (qPCR) for tuberculosis detection in paraffin-embedded tissues.The evaluation criteria included sensitivity, specificity, positive predictive value, negative predictive value, and accuracy rate. RESULTS: Based on the clinical diagnostic results, the EasyNAT assay demonstrated a sensitivity of 87.1%(88/101, 95%CI: 79.2%-92.3%)and a specificity of 100.0%(33/33, 95%CI: 89.6%-100.0%).The positive predictive value, negative predictive value, and accuracy rate were 100% (88/88, 95%CI: 95.8%-100.0%), 71.7%(33/46, 95%CI: 57.5%-82.7%), and 90.3%(121/134, 95%CI: 84.1%-94.2%), respectively.In comparison, the qPCR assay exhibited a sensitivity of 96.0%(90.3%-98.5%)and a specificity of 100.0%(89.6%-100.0%).The positive predictive value, negative predictive value, and accuracy rate for qPCR were 100.0%(96.2%-100.0%), 89.2%(75.3%- 95.7%), and 97.0%(92.6%-98.8%).The Cohen's kappa value of 0.84 indicated substantial agreement between EasyNAT and qPCR.The detection rate of tuberculosis using this method was 86.4%(38/44, 95%CI: 73.3%-93.6%), while the detection rate for extrapulmonary tuberculosis was 87.7%(50/57, 95%CI: 76.8%-93.9%).In comparison, qPCR showed a detection rate of 97.7%(88.2%- 99.6%) for pulmonary tuberculosis and 94.7%(85.6%-98.6%)for extrapulmonary tuberculosis.There was no statistically significant difference in the detection results between the method and qPCR for both pulmonary and extrapulmonary tuberculosis(P>0.05).Importantly, the EasyNAT detection combined nucleic acid extraction, amplification, and analysis into one process.Compared with traditional qPCR methods, manual operation time was reduced by 2 hours, leading to an overall reduction in total testing time by 3 hours. CONCLUSION The EasyNAT nucleic acid rapid detection system can quickly, conveniently, and accurately detect Mycobacterium tuberculosis DNA in paraffin-embedded tissues, demonstrating significant clinical utility in the pathological diagnosis of tuberculosis.
Humans ; Retrospective Studies ; Paraffin Embedding ; Sensitivity and Specificity ; Tuberculosis/microbiology* ; Real-Time Polymerase Chain Reaction ; Mycobacterium tuberculosis/genetics* ; Predictive Value of Tests ; Nucleic Acid Amplification Techniques/methods* ; Female ; Male

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*

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

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