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*

Rhythm, as a prominent characteristic of auditory experiences such as speech and music, is known to facilitate attention, yet its contribution to working memory (WM) remains unclear. Here, human participants temporarily retained a 12-tone sequence presented rhythmically or arrhythmically in WM and performed a pitch change-detection task. Behaviorally, while having comparable accuracy, rhythmic tone sequences showed a faster response time and lower response boundaries in decision-making. Electroencephalographic recordings revealed that rhythmic sequences elicited enhanced non-phase-locked beta-band (16 Hz-33 Hz) and theta-band (3 Hz-5 Hz) neural oscillations during sensory encoding and WM retention periods, respectively. Importantly, the two-stage neural signatures were correlated with each other and contributed to behavior. As beta-band and theta-band oscillations denote the engagement of motor systems and WM maintenance, respectively, our findings imply that rhythm facilitates auditory WM through intricate oscillation-based interactions between the motor and auditory systems that facilitate predictive attention to auditory sequences.
Humans ; Memory, Short-Term/physiology* ; Male ; Beta Rhythm/physiology* ; Female ; Theta Rhythm/physiology* ; Young Adult ; Auditory Perception/physiology* ; Adult ; Electroencephalography ; Acoustic Stimulation ; Reaction Time/physiology* ; Brain/physiology* ; Attention/physiology*

Attention is the cornerstone of effective functioning in a complex and information-rich world. While the neural activity of attention has been extensively studied in the cortex, the brain-wide neural activity patterns are largely unknown. In this study, we conducted a comprehensive analysis of neural activity across the mouse brain during attentional processing using EEG and c-Fos staining, utilizing hierarchical clustering and c-Fos-based functional network analysis to evaluate the c-Fos activation patterns. Our findings reveal that a wide range of brain regions are activated, notably in the high-order cortex, thalamus, and brain stem regions involved in advanced cognition and arousal regulation, with the central lateral nucleus of the thalamus as a strong hub, suggesting the crucial role of the thalamus in attention control. These results provide valuable insights into the neural network mechanisms underlying attention, offering a foundation for formulating functional hypotheses and conducting circuit-level testing.
Animals ; Attention/physiology* ; Mice ; Brain/physiology* ; Male ; Electroencephalography ; Reaction Time/physiology* ; Brain Mapping ; Mice, Inbred C57BL ; Choice Behavior/physiology* ; Proto-Oncogene Proteins c-fos/metabolism*

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

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

Quantitative real-time polymerase chain reaction (qPCR) is one of the most widely used molecular pathological diagnostic techniques in China due to its advantages of the simple operation, short turnaround time, high sensitivity, and standardizable result analysis. However, in clinical practice, there is not yet an expert consensus to guide molecular pathological diagnostics of tumor using qPCR techniques in terms of validation and verification of method performance, quality management and interpretation of complex results. Therefore, this expert consensus aims to provide standardized opinions on the practical application of qPCR techniques, and suggestions on how to deal with common problems and abnormal results, and reach a Chinese expert consensus on the clinical practice of qPCR techniques in molecular pathological diagnostics of tumor, in order to standardize the testing process, improve the accuracy of results, and promote the clinical applications of qPCR techniques.
Humans ; Real-Time Polymerase Chain Reaction ; Consensus ; East Asian People ; Neoplasms ; China

The secondary motor cortex (M2) encodes choice-related information and plays an important role in cue-guided actions. M2 neurons innervate the dorsal striatum (DS), which also contributes to decision-making behavior, yet how M2 modulates signals in the DS to influence perceptual decision-making is unclear. Using mice performing a visual Go/No-Go task, we showed that inactivating M2 projections to the DS impaired performance by increasing the false alarm (FA) rate to the reward-irrelevant No-Go stimulus. The choice signal of M2 neurons correlated with behavioral performance, and the inactivation of M2 neurons projecting to the DS reduced the choice signal in the DS. By measuring and manipulating the responses of direct or indirect pathway striatal neurons defined by M2 inputs, we found that the indirect pathway neurons exhibited a shorter response latency to the No-Go stimulus, and inactivating their early responses increased the FA rate. These results demonstrate that the M2-to-DS pathway is crucial for suppressing inappropriate responses in perceptual decision behavior.
Mice ; Animals ; Motor Cortex ; Corpus Striatum/physiology* ; Neostriatum ; Neurons/physiology* ; Reaction Time

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 analyze the genetic characteristics of varicella-zoster virus (VZV) in people aged 20 years and under in Yichang City of Hubei Province from 2019 to 2020. Methods: Based on the Yichang Health Big Data Platform, we investigated cases 20 and under clinically diagnosed as herpes zoster in three hospitals from March 2019 to September 2020. Collecting vesicle fluid and throat swab samples of the cases and completing questionnaires to obtain basic information. Real-time fluorescent quantitative PCR was used for positive identification of the virus. PCR amplification of VZV's open reading frame (ORF) and sequencing of the products to determine the VZV genotype. Analyze mutations at some specific single nucleotide polymorphism (SNP) sites. Results: Among 46 cases of herpes zoster, the male to female ratio was 1.3∶1 (26∶20) and the age ranged from 7 to 20 years old. Fifteen cases had been vaccinated against varicella, including 13 and 2 cases of 1 and 2 doses, respectively. VZV strains were detected in 34 samples (73.91%), all belonging to Clade 2. Phylogenetic tree analysis of the nucleotide of ORF22 showed, compared with Clade 2 referenced strains, the sequence matching degree of nucleotide for all 34 samples was 99.0% to 100.0%. Conclusion: The main VZV strain causing herpes zoster in people aged 20 years and under in Yichang from 2019 to 2020 was Clade 2.
Humans ; Child ; Adolescent ; Young Adult ; Adult ; Herpesvirus 3, Human/genetics* ; Phylogeny ; Herpes Zoster/epidemiology* ; Polymorphism, Single Nucleotide ; Real-Time Polymerase Chain Reaction ; Nucleotides

Objective: To establish and optimize a TaqMan-probe quantitative real-time PCR (qPCR) assay for the detection of 7 important Rickettsiales pathogens and simultaneous identification of the infection types. Methods: Based on the ompB gene of Rickettsia prowazekii, Rickettsia mooseri and spotted fever group rickettsiae, the groEL gene of Orientia tsutsugamushi, the 16S rRNA of Ehrlichia chaffeensis, the gltA gene of Anaplasma phagocytophilum and the com1 gene of Coxiella burnetii, we synthesized primers and TaqMan-probes and optimized the reaction system and reaction process to same solution. The sensitivity, specificity and reproducibility of this assay were evaluated and the assay was used for the detection of simulated and actual samples. Results: The Ct value of the standard curves of the 7 pathogens showed a good linear relationship with the number of DNA copies (all R² >0.990 0), the minimum detection limit was 10 copies/μl, showing good specificity. In the 96 tick nucleic acid extracts, Coxiella burnetii was detected in 1 sampleand spotted fever group Rickettsiae was detected in 3 samples. In the 80 blood samples from patients with undefined febrile illness, Orientia tsutsugamushi was detected in 1 sample and spotted fever group rickettsiae was detected in 2 samples. Conclusions: In this study, based on the established TaqMan-probe qPCR assay, the reaction system and reaction condition of the 7 important pathogens of Rickettsiales were optimized to the same solution. This method overcomes the shortcomings of using different reaction systems and reaction conditions for different pathogens, which can precisely identify the species of 7 important pathogens of Rickettsiales in clinical sample detections and is important for the infection type identification and laboratory detection time reduction to facilitate precise treatment of the patients.
Humans ; Rickettsiales ; Real-Time Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; Reproducibility of Results ; Orientia tsutsugamushi ; Spotted Fever Group Rickettsiosis