The emergence of clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) system represents a revolutionary paradigm shift in molecular diagnostics, offering transformative potential for RNA analysis within the rigorous demands of forensic science. Conventional forensic RNA detection methodologies, such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or microarray analysis, are significantly hampered by inherent limitations including complex, multi-step protocols requiring sophisticated laboratory infrastructure, pronounced susceptibility to inhibitors prevalent in complex forensic matrices (e.g., humic acids, heme, indigo dyes), and often inadequate sensitivity for trace or degraded samples typical of crime scenes, thereby failing to meet the critical operational imperatives of forensic practice: rapidity, high specificity, sensitivity, portability, and robustness against interference. This review posits that CRISPR-Cas-based RNA detection technology provides a groundbreaking solution by leveraging the programmable, sequence-specific recognition conferred by the synergistic interaction between a designed guide RNA (gRNA) and Cas effector proteins (e.g., Cas12a, Cas13a, Cas14). Upon target RNA binding, specific Cas enzymes undergo conformational activation, exhibiting collateral cleavage activity―a unique catalytic amplification mechanism where the enzyme non-specifically cleaves surrounding reporter molecules, enabling ultra-high sensitivity. To further enhance detection limits, CRISPR-Cas systems are strategically integrated with isothermal pre-amplification techniques like recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP), which efficiently amplify target RNA at constant temperatures, eliminating the need for thermal cyclers. This powerful cascade―isothermal pre-amplification followed by CRISPR-mediated sequence-specific recognition and collateral signal amplification―achieves exceptional sensitivity, often down to the single-molecule (attomolar) level, while drastically reducing analysis time to potentially 30-60 min. Crucially, the compatibility of CRISPR-Cas detection with simple, equipment-free readout systems, such as lateral flow strips (LFS) for visual colorimetric results or portable fluorescence/electrochemical sensors, facilitates true point-of-need (PON) forensic analysis directly at crime scenes, morgues, or field labs. This enables rapid applications like specific body fluid identification (e.g., distinguishing menstrual blood via miRNA, identifying saliva via mRNA), post-mortem interval (PMI) estimation through RNA degradation/expression patterns, donor age inference via age-related RNA markers, tissue identification, and microbial forensics, thereby accelerating investigative leads, minimizing sample degradation risks, and optimizing resource allocation. However, significant challenges impede widespread adoption, including persistent environmental interference inhibiting enzymes, fluctuations in Cas/amplification enzyme activity affecting reproducibility, a critical lack of standardized protocols and validated quality assurance/quality control (QA/QC) frameworks essential for forensic reliability and court admissibility, and current limitations in multiplex detection capability. Consequently, future research must prioritize overcoming multiplexing bottlenecks for comprehensive analysis, enhancing system robustness through Cas protein engineering and optimized reagents, developing fully integrated, sample-to-answer microfluidic or lateral flow devices for user-friendly field deployment, and collaboratively establishing universally accepted validation guidelines, performance standards, and stringent QA/QC procedures. Furthermore, the urgent development of clear ethical guidelines governing the use of this highly sensitive technology, particularly concerning RNA data privacy and potential misuse, is imperative. This review systematically outlines the principles, forensic applications, current limitations, and future trajectories of CRISPR-RNA detection, with the authors’ conviction that focused efforts addressing these challenges will translate this technology into a cornerstone of next-generation forensic practice, driving unprecedented efficiency and innovation in field investigations and laboratory analysis to enhance justice delivery.

Objective To explore the clinical efficacy and safety of one-stage posterior lesion removal and internal spinal fixation in patients with lumbar Brucellosis spondylitis.Methods The clinical data of 24 patients admitted from October 2017 to October 2022 were retrospectively analyzed,2 patients were lost to follow-up at 10 months after surgery,at the final 22 cases were included in the study,including 13 males and 9 females with an average age of(52.00±6.89)years old,were treated with one-stage posterior lesion removal and internal spinal fixation.The operation time,intraoperative bleeding,follow-up time,ery-throcyte sedimentation rate(ESR)and C-reactive protein(CRP)before and after operation were recorded.The pain visual ana-logue scale(VAS),Oswestry disability index(ODI),the Japanese Orthopaedic Association(JOA)score for neurofunction,American Spinal Injury Association(ASIA)spinal cord injury grade and modified MacNab criteria were ussed to evaluate the efficacy.Results All patients were followed up from 12 to 30 months with an average of(17.41±4.45)months.The operation time was 70 to 155 min with an average of(1 16.59±24.32)min;the intraoperative bleeding volume was 120 to 520 ml with an average of(275.00±97.53)ml.CRP and ESR levels decreased more significantly at 1 week and at the final follow-up than pre-operative levels(P＜0.05).VAS,JOA score and ODI at 1 week and at the latest follow-up were more significantly improved than preoperative results(P＜0.05).There was no significant difference between ASIA preoperative and 1 week after operation(P＞0.05),and a significant difference between preoperative and last follow-up(P＜0.05).In the final follow-up,21 patients had ex-cellent efficacy,1 patient had fair,and there was no recurrence during the follow-up.Conclusion One-stage transpedicular le-sion removal and internal spinal fixation,with few incisions and short operation time,helps the recovery of neurological func-tion,and the prognosis meets the clinical requirements,which can effectively control Brucella spondylitis.

On the basis of the laboratory detection results of influenza viruses in Jiangsu Province from 2022 to 2023,this study was aimed at conducting a analysis of the genetic characteristics and resistance of the H3N2 influenza virus,and evalua-ting the effectiveness of vaccines and drugs.The full genome of 29 H3N2 isolates submitted by provincewide influenza surveil-lance network laboratories was amplified and sequenced.Phylogenetic trees of the HA and NA genes were constructed.The mutation characteristics of antigenic sites,glycosylation sites,and resistance sites were analyzed,and the influenza virus neura-minidase was detected with a fluorescence luminescence method.The homology of the 29 H3N2 isolates in Jiangsu Province was relatively high.The isolates belonged to the same evolutionary branch as domestic reference strains and the 2021-2022 vaccine strains,but were in different evolutionary branches from foreign reference strains and the 2022-2023 vaccine strains.Compared with the 2021-2022 vaccine strain A/Cambodia/e0826360/2020,the H3N2 influenza viruses isolated in Jiangsu Province during 2022-2023 had four amino acid substitutions in the HA protein and two amino acid substitutions in the NA protein.No changes in glycosylation sites,receptor-binding sites,and conserved amino acid residue regions were observed.IC50 analysis indicated that the 29 H3N2 isolates were not resistant to the antiviral drugs oseltamivir and zanamivir.The pre-dominant influenza viruses detected in Jiangsu Province during 2022-2023 were type A H3N2 influenza viruses.Monitoring of variations in HA and NA gene sites and influenza virus resistance aids in rapid understanding gene mutations,and can inform the selection of effective vaccine strains,and contribute to the prevention and control of influenza virus outbreaks.

AIM To investigate the effects of stir-frying,processing with butter and carbonizing by stir-frying on oil components in Gleditsiae sinensis Fructus and Gleditsiae Fructus Abnormalis.METHODS The volatile oils and fatty oils were extracted by steam distillation method and Soxhlet extraction method,respectively,after which the extraction rates were determined.GC-MS was applied to analyzing the kinds and relative contents of oil components,after which cluster analysis was performed.RESULTS After the processing,the two medicinal materials demonstrated increased extraction rates of fatty oils and decreased extraction rates of volatile oils(except for processing with butter),the extraction rates of oil components in Gleditsiae sinensis Fructus were higher than those in Gleditsiae Fructus Abnormalis,and the reduced relative contents of toxic olefin benzene components were observable.CONCLUSION The kinds and relative contents of oil components in Gleditsiae sinensis Fructus and Gleditsiae Fructus Abnormalis exist obvious differences,the former displays better medicinal quality,whose processing mechanism in alleviating dryness and strength may contribute to the reduction of relative contents of toxic olefin benzene components.

AIM To establish an HPLC method for the simultaneous content determination of 3′-hydroxy-puerarin,hydroxysafflor yellow A,puerarin,puerarin apigenin,3′-methoxypuerarin,daidzein,stilbene glycoside,luteolin side,genistein side,luteolin B and rosmarinic acid in Naomaitai Capsules.METHODS The analysis was performed on a 30℃ thermostatic Kromasil C18 column(250 mm×4.6 mm,5 μm),with the mobile phase comprising of acetonitrile-water(containing 1.0 g/L phosphoric acid)flowing at 0.8 mL/min in a gradient elution manner,and the detection wavelength was set at 280 nm.Subsequently,principal component analysis and cluster analysis were made.RESULTS Twelve components showed good linear relationships within their own ranges(r＞0.999 0),whose average recoveries were 97.00%-99.00% with the RSDs of 0.86%-1.82% .Ten batches of samples were clustered into four categories,the accumulative variance contribution rate of four principal components reached 86.262% .CONCLUSION This simple,stable,reliable and accurate method can be used for the quality control of Naomaitai Capsules.

Objective:To develop an integrated point-of-care testing (POCT) reagent for genotying respiratory syncytial virus (RSV) and evaluate its performance.Methods:Specific primers and probes were designed based on the conserved sequences of the genomes of RSV A and B as well as ON1 and BA9 genotypes. The PCR reaction system and conditions were optimized. The vitrification technology of reagents and multiplex detection platform were integrated to develop the RSV genotyping POCT reagent. The sensitivity, specificity, reproducibility, and clinical performance of the product were then evaluated.Results:The sensitivity of the developed integrated RSV genotyping POCT reagent reached 500 copies/ml. It exhibited good specificity with no cross-reaction with clinically similar pathogens. The coefficient of variation of Ct values for both inter-batch and intra-batch reproducibility was less than 5%, indicating good reproducibility. In testing 53 clinical samples, the detection results showed high consistency and concordance with the reference reagent, with a positive concordance rate of up to 98.11%.Conclusions:The developed integrated RSV genotyping POCT reagent incorporates nucleic acid extraction, purification, and detection into a single process, achieving a "sample in, result out" workflow. It is simple to operate and provides accurate, reliable, and stable detection results. This product can be used for the genotyping of RSV A and B in POCT, offering support for the prevention, control, and diagnosis of RSV.

Objective To explore the current status of H.pylori infection in the natural population of Sanya City,analyze its influencing factors,and provide a reference basis for the prevention and control of H.pylori infection.Methods A total of 677 residents from four districts of Sanya City were selected by overall stratified random sampling method,and were subjected to urea 14C breath test and questionnaire survey to calculate the positive rate of H.pylori in the natural population and analyze the influencing factors of H.pylori infection.Results A total of 606 residents were included,and the number of H.pylori positive detections was 261,with a positive detection rate of 38.5％.Among them,different ethnicity,marital status,smoking,eating vegetables and fruits,and literacy level were associated with H.pylori infection(P＜0.05);gender,age,BMI,alcohol consumption,drinking water source,betel quid chewing,and the number of cohabitants were not significantly associated with H.pylori infection(P＞0.05).Family infection was an independent risk factor for H.pylori infection in the natural population of Sanya City,and Li ethnicity,frequent consumption of fruits and vegetables,and college and higher education level were independent protective factors for H.pylori infection in the natural population of Sanya City.Conclusion The rate of H.pylori infection in the natural population of Sanya City is lower than the national average.Consuming more fruits and vegetables and improving the awareness of hygiene protection are conducive to the prevention of H.pylori infection;and the promotion of the family and related members with the same examination and treatment is important to avoid aggregation of infection within the family.

Bone marrow biopsy is one of the important means of hematopathological diagnosis, which has decisive diagnostic significance for various benign and malignant lymphohematopoietic system diseases. Its diagnostic value includes morphological observation, immunohistochemistry, genetics, and molecular biology testing. Owing to the unique nature of bone marrow biopsy, decalcification is an essential step in the pre-treatment process. Its purpose is to remove calcium from bone tissue, preserve intact collagen fiber components, facilitate tissue sectioning, and prevent tissue detachment during staining. If bone marrow biopsy lacks sufficient decalcification, preparing a section is difficult. Conversely, if decalcification is excessive, it can seriously disrupt tissue antigen activity. Therefore, a decalcification method with high decalcification efficiency and mild antigen damage is essential for bone marrow biopsy. This article introduces a bone marrow biopsy tissue decalcification method with high efficiency and less antigen loss: decalcification is performed at room temperature with 12% formic acid and 8% hydrochloric acid decalcification solution on a shaker.

Objective To investigate the radiotherapy dose impacts of different jaw widths on stereotactic body radiotherapy(SBRT)for hepatocellular carcinoma(HCC)with the hilical tomotherapy(HT)planning system.Methods Totally 16 HCC patients who received radiotherapy at some hospital from March 2021 to August 2023 were selected,and the planning was carried out with the same pitch,modulation factors and optimization conditions and the jaw widths being 1.0,2.5 and 5.0 cm.The dosimetric differences due to the jaw widths in planned targets and organs at risk(OAR)were compared,and the planned treatment time,monitor unit,gantry rotations and gantry period were evaluated.SPSS 22.0 was used for statistical analysis.Results Better dosimetric parameters and lower doses to OARs could be got with lower jaw widths.The widened jaw widths resulted in reduced planned treatment time,decreased monitor units and gantry rotations,shortened gantry period while enhanced treatment efficiency.Conclusion A 2.5 cm jaw width for HT planning contributes to improving treatment efficiency during HCC SBRT under the premise of ensured target dose distribution and satisfactory dose to OAR for clinical require-ments.[Chinese Medical Equipment Journal,2024,45(7):51-55]

Objective To investigate the risk factors for bronchopulmonary dysplasia(BPD)in twin preterm infants with a gestational age of<34 weeks,and to provide a basis for early identification of BPD in twin preterm infants in clinical practice.Methods A retrospective analysis was performed for the twin preterm infants with a gestational age of<34 weeks who were admitted to 22 hospitals nationwide from January 2018 to December 2020.According to their conditions,they were divided into group A(both twins had BPD),group B(only one twin had BPD),and group C(neither twin had BPD).The risk factors for BPD in twin preterm infants were analyzed.Further analysis was conducted on group B to investigate the postnatal risk factors for BPD within twins.Results A total of 904 pairs of twins with a gestational age of<34 weeks were included in this study.The multivariate logistic regression analysis showed that compared with group C,birth weight discordance of>25%between the twins was an independent risk factor for BPD in one of the twins(OR=3.370,95%CI:1.500-7.568,P<0.05),and high gestational age at birth was a protective factor against BPD(P<0.05).The conditional logistic regression analysis of group B showed that small-for-gestational-age(SGA)birth was an independent risk factor for BPD in individual twins(OR=5.017,95%CI:1.040-24.190,P<0.05).Conclusions The development of BPD in twin preterm infants is associated with gestational age,birth weight discordance between the twins,and SGA birth.