BackgroundMetabolic dysfunction-associated steatohepatitis (MASH), the progressive phenotype of metabolic dysfunction-associated steatotic liver disease (MASLD), is well recognized for its increased risk of progressing to cirrhosis and hepatocellular carcinoma (HCC). However, early diagnosis and identification of the MASH patients with a tendency toward malignancy HCC remain a significant challenge.MethodIn previous studies, we established a novel conditional inducible HRAS gene expression murine model with normal diet and lifestyle conditions, which exhibited 100% HCC incidence and recapitulated the four major progression stages of MASH to HCC: MASH, fibrosis, cirrhosis, and HCC. Based on this model, we revealed the potential markers of MASH prone to HCC characteristics of malignant MASH through RNA-Seq and bioinformatics analysis, and further confirmed by pathological biopsy, biochemical tests, inflammatory cytokines measurement, Oil O red staining, and immunohistochemical examination.ResultsFor MASH, the initial stage of HCC, we observed evidence of hyperlipidemia and insulin resistance in the HRAS mouse model based on blood morphology, biochemical parameters, and insulin tolerance test. Furthermore, the pathological features of MASH were confirmed by the presence of hepatocellular fatty vacuolar changes, lipid droplet accumulation, and inflammation. Then, RNA-Seq analysis revealed the molecular signatures of malignant MASH and revealed three novel potential markers associated with adverse progression of MASH to HCC: Klk1b4, Alox5, and Pla2g2e.ConclusionBased on a stable and novel murine model of MASH prone to HCC, we, for the first time, presented a comprehensive molecular signature and identified novel potential adverse progression markers of MASH prone to malignant HCC, which contributed possibly to early clinical diagnosis and prognostic assessment, even becoming potential therapeutic target.

A multiplex liquid array assay based on Luminex xTAG technology was developed for the first time for the simultaneous detection of five respiratory bacterial pathogens in Specific Pathogen-Free (SPF) chickens—Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), Pasteurella multocida (Pm), Avibacterium paragallinarum (Apg), and Mycobacterium avium (Mav)—filling the gap that these five pathogens could not be simultaneously detected previously. Specific primers were designed targeting conserved genes of each pathogen, with a TAG sequence attached to the 5′ end of each forward primer and biotin to the 5′ end of each reverse primer. After multiplex PCR amplification, the products were hybridized with MagPlex-TAG microspheres and analyzed using the Luminex system. The method was validated for sensitivity, specificity, and repeatability, and tested with simulated positive samples (a mixture of genomic DNA from the five target pathogens) and SPF chicken samples. The LOD was 10 copies/μL for Pm and 100 copies/μL for the other four pathogens. No cross-reactivity was observed with 18 non-target bacterial species. Intra- and Inter-assay coefficients of variation (CV) ranged from 0.8% to 12.2% and 2.3% to 14.4%, respectively, both below 15%. All simulated mixed positive samples yielded results consistent with expectations, and none of the 114 nasopharyngeal swab samples from SPF chickens tested positive for any of the five targets. The developed assay offers high throughput, high sensitivity, high specificity, and good repeatability, providing a robust tool for routine quality control in SPF chickens.

Cryptosporidium parvum (Cp), Strongyloides stercoralis (Ss), Trypanosoma cruzi (Tc), Theileria ovis (To) and Echinococcus granulosus (Eg) are significant in causing similar clinical symptoms in both humans and animals. These symptoms include diarrhea, fever, pneumonia, hepatitis, encephalitis, cardiovascular, and gastrointestinal diseases, as well as reproductive system diseases. Therefore, it is urgent to establish a rapid, specific, and sensitive method to simultaneously detect these five pathogens. Here, we develop a TaqMan probe-based pentaplex real-time fluorescence quantitative PCR (qPCR) for simultaneous detection of Cp, Ss, Tc, To, and Eg for the first time. Five sets of specific primers and probes were designed. The 5' end of five probes were labeled with five different fluorescent groups (FAM, VIC, ABY, JUN, CY5) that do not interfere with each other, while the 3' end were labeled with quenching groups (MGB-NFQ, QSY). The instrument collects fluorescence signals at different wavelengths in five independent optical channels in qPCR to achieve simultaneous specific amplification and quantitative detection of five targets within one tube. A five-color qPCR was developed, which can rapidly and quantitatively distinguish and identify Cp, Ss, Tc, To, and Eg in a single tube. This method shows strong specificity and no cross-reaction with other control pathogens. This method has high sensitivity, with the LLOQ of 10 copies and the LOD as low as 1 copy. The standard curve shows a good linear relationship. The amplification efficiency is high. The repeatability and reproducibility are excellent. The interference test shows that high concentrations of nucleic acids do not interfere with low concentrations of nucleic acids, and can ensure excellent detection results even in complex samples for 1.5 hours. A total of 246 clinical samples were detected: 4.88% were positive for Cp, 2.44% for Ss, and 15.85% for To, while 0.81% for co-infections of Cp/To. This TaqMan probe-based pentaplex qPCR enables rapid detection and differential identification of five pathogens from clinical samples, eliminating the need for multiple single-plex qPCR. It can potentially assist clinicians in diagnosis and treatment planning by rapidly detecting pathogens in clinical samples and further quantitative analysis. It is of extremely significant importance for protecting human health.

Pneumocystis is an opportunistic fungal pathogen that causes severe Pneumocystis pneumonia (PCP) in immunocompromised individuals and laboratory animals. Three host-specific species—Pneumocystis murina (P. murina), Pneumocystis carinii (P. carinii), and Pneumocystis jirovecii (P. jirovecii)—are closely associated with infections in humans and laboratory animals. However, the conventional method, microscopic staining, suffers from low sensitivity, operator-dependent subjectivity, and inability to differentiate species, highlighting the urgent need for a multiplex qPCR assay. In this study, we established a multiplex qPCR method targeting the mtLSUrRNA gene of P. murina, the TS gene of P. carinii, and the mtSSUrRNA gene of P. jirovecii. Primers and probes were designed and optimized using a matrix approach. The method was systematically evaluated for sensitivity, specificity, and reproducibility using recombinant plasmid standards and laboratory animal samples. Validation was performed on 260 mouse lung samples, 30 P. murina-positive samples, 25 rat lung samples, 6 rat bronchoalveolar lavage fluid (BALF) samples, and 8 P. carinii-positive samples. Results were compared with single-plex qPCR and staining microscopy (performed on 68 mouse lung samples, 38 Pneumocystis-positive samples). The limits of detection (LOD) were 5 copies/μL for P. murina, 6 copies/μL for P. carinii, and 8 copies/μL for P. jirovecii. Standard curves showed excellent linearity (R2 ≥ 0.999) with amplification efficiencies of 90–110%. No non-specific reactions were observed with 22 common pathogens, and intra-/inter-group coefficients of variation (CV%) were below 1%. Moreover, interference testing revealed minimal matrix effects on the amplification system and no mutual interference among the primers and probes. The multiplex qPCR detected all 38 positive samples (100%), showing 100% concordance with single-plex qPCR, whereas Giemsa staining detected none (0%) and toluidine blue staining only 60% (3/5) of the tested positives, suggesting that the multiplex qPCR achieved higher detection rates than staining microscopy. In conclusion, this novel multiplex qPCR method offers high sensitivity, specificity, and reproducibility, providing a sensitive and specific tool for laboratory animal health monitoring and epidemiological surveillance. Its clinical application for human PCP diagnosis requires further validation with authentic human specimens.

Pre-exposure prophylaxis of HIV infection with a microbicide is one of the most effective ways to block HIV/AIDS transmission. In this study, a potent and long-acting HIV entry inhibitor peptide 2P23 was developed as a novel HIV microbicide, and its toxicity and safety were evaluated through animal model in vivo for the first time, which provided a new effective means for the prevention of HIV/AIDS. In order to evaluate the potential effect to cause human toxicity and possible health hazards likely to arise from repeated rectovaginal application of 2P23, we conducted an acute toxicity in which a dose of 300 mg/kg of 2P23 was administrated by rectovaginal three times within 24-hour in male and female Sprague Dawley rats. Furthermore, a 90-day repeated rectovaginal treatment tests in rats were used to evaluate the chronic and reproductive toxicity of 50, 100, 200 mg/kg of 2P23. The effects of 2P23 administration on morbidity, mortality, clinical signs, changes in body weight, food consumption, clinical pathology, organ weight, hematological and physiological biochemical parameter indicators, histopathology, changes in rectovaginal microbiota, fertility, and early embryonic development were assessed. There were no mortality or abnormal clinical signs, no significant changes in body weight gain or hematological and physiological biochemical parameter indicators, no changes in necropsy and histopathology findings of vital organs, and no significant changes in rectum and vagina microbiota associated with the administration of 2P23. There were no effects on fertility and early embryonic development in male and female rats. The results indicate that 2P23 does not cause significant adverse effects in the 90-day and suggest that the no observed adverse effect level is 300 mg. This study has important application value and good innovation for the development of anti-HIV agents for rectovaginal administration. The novel HIV microbicide is safe and effective for the prevention and control of HIV/AIDS sexually transmitted infections, which enriches people's understanding of the novel HIV microbicide gel.

The emergence of coinfection with Toxoplasma gondii, Neospora caninum, Eimeria stiedai, Giardia lamblia, and Trypanosoma evansi is an important problem that endangers human health, animal quality and public sanitary security. These pathogenic protozoans play important roles in establishment of similar clinical signs of diseases in humans, pigs, sheep, and rabbits, including fever, diarrhea, hepatitis, encephalitis, and reproductive disorders. Therefore, a rapid and specific diagnostic method to simultaneously detect these five pathogens is urgently required. Here, we developed a TaqMan-probe-based quantitative real-time polymerase chain reaction (qPCR) for the simultaneous detection of these five pathogens for the first time. Specific primers and probes were designed targeting the G3PDH gene of Toxoplasma gondii, the NC5 gene of Neospora caninum, the ADF gene of Eimeria stiedai, the GDH gene of Giardia lamblia, the COX1 gene of Trypanosoma evansi, and a TaqMan-probe-based pentaplex qPCR assay capable of simultaneously detecting these five pathogens was developed. The assay showed strong specificity, with no cross-reactivity detected against nucleic acids from other control pathogens. The assay demonstrated high sensitivity, with the lower limit of quantification (LLOQ) of 10 copies per reaction for the recombinant plasmid standards pTgG3PDH, pNcNC5, pEsADF, pGlGDH, pTeCOX1, and the limit of detection (LOD) as low as 1.1 copies. The standard curves exhibited excellent linearity (correlation coefficient values of 0.996, 0.996, 0.996, 0.992, and 0.996, respectively) and high amplification efficiencies (95.534%, 96.203%, 107.818%, 100.851%, and 104.487%, respectively). The assay also exhibited excellent repeatability and reproducibility, with inter- and intra-assay coefficient of variation (CV) ranging from 0.07% to 2.13%. The anti-interference test showed that high-concentration nucleic acids did not interfere with low-concentration nucleic acids, thus ensuring excellent detection results even in complex samples. Among 210 clinical samples, the assay detected Toxoplasma gondii in 2.38%, Neospora caninum in 2.38%, Eimeria stiedai in 10%, and Giardia lamblia in 36.19%. The coinfection rates were 8.1% for Eimeria stiedai & Giardia lamblia. This TaqMan-probe-based pentaplex qPCR assay offers a rapid, sensitive, and specific tool for the simultaneous detection of Toxoplasma gondii, Neospora caninum, Eimeria stiedai, Giardia lamblia, and Trypanosoma. It is of great significance to safeguard human health.

Dengue virus (DENV) infection remains a critical global health threat, with DENV-2 being the most virulent serotype capable of causing lethal dengue hemorrhagic fever (DHF), a severe complication characterized by plasma leakage and hemorrhagic manifestations. While the search for viral receptors and immunocompetent animal models has persisted since the first recorded outbreak in 1779, significant gaps remain. Here, we establish the first immunocompetent murine model of DHF with intact innate/adaptive immunity by generating hTim4-transgenic C57BL/6J mice. This model recapitulates fatal DHF complications seen in humans, including systemic hemorrhage, dengue encephalitis and intestinal ischemia/gangrene. Integrated single-cell RNA sequencing and spatial transcriptomics analysis of hemorrhagic gut lesions demonstrated that DENV-2 infection induces Syk protein overexpression, leading to enhanced Th2 cytokine secretion and impaired hemostatic regulation. This cascade enhances vascular permeability, promotes plasma leakage, and drives multiorgan hemorrhage, a mechanism corroborated by parallel analyses of human DHF tissues. Critically, Th2-biased cytokine storm mirrors clinical findings in severe human dengue cases. Our work not only identifies hTim4 as a functional DENV-2 receptor but also provides a mechanistically grounded platform for DHF pathogenesis studies, bridging critical gaps between preclinical models and human immunopathology.

Abstract Background Pigeon circovirus infections in pigeons (Columba livia domestica) have been reported worldwide. Pigeons should be PiCV-free when utilized as qualified experimental animals. However, pigeons can be freely purchased as experimental animals without any clear guidelines to follow. Herein, we investigated the status quo of PiCV infections on a pigeon farm in Beijing, China, which provides pigeons for experimental use. Results PiCV infection was verified in at least three types of tissues in all forty pigeons tested. A total of 29 full-length genomes were obtained and deposited in GenBank. The whole genome sequence comparison among the 29 identified PiCV strains revealed nucleotide homologies of 85.8–100%, and these sequences exhibited nucleotide homologies of 82.7–98.9% as compared with those of the reference sequences. The cap gene displayed genetic diversity, with a wide range of amino acid homologies ranging from 64.5% to 100%. Phylogenetic analysis of the 29 full-genome sequences revealed that the PiCV strains in this study could be further divided into four clades: A (17.2%), B (10.4%), C (37.9%) and D (34.5%). Thirteen recombination events were also detected in 18 out of the 29 PiCV genomes obtained in this study. Phylogenetic research using the rep and cap genes verified the recombination events, which occurred between clades A/F, A/B, C/D, and B/D among the 18 PiCV strains studied. Conclusions In conclusion, PiCV infection, which is highly genetically varied, is extremely widespread on pigeon farms in Beijing. These findings indicate that if pigeons are to be used as experimental animals, it is necessary to evaluate the impact of PiCV infection on the results.

Helicobacter pylori ( H. pylori, Hp ) has been designated a class I carcinogen and is closely associated with severe gastric diseases. During colonization in the gastric mucosa, H. pylori develops immune escape by inducing host immune tolerance. The gastric epithelium acts as the first line of defense against H. pylori , with Toll-like receptors (TLRs) in gastric epithelial cells being sensitive to H. pylori components and subsequently activating the innate immune system. However, the mechanism of immune tolerance induced by H. pylori through the TLR signalling pathway has not been fully elucidated. In this research, we detected the expression of TLRs and inflammatory cytokines in GES-1 cells upon sustained exposure to H. pylori or H. pylori lysate from 1 to 30 generations and in Mongolian gerbils infected with H. pylori for 5 to 90 weeks. We found that the levels of TLR6 and inflammatory cytokines first increased and then dropped during the course of H. pylori treatment in vitro and in vivo. The restoration of TLR6 potentiated the expression of IL-1β and IL-8 in GES-1 cells, which recruited neutrophils and reduced the colonization of H. pylori in the gastric mucosa of gerbils. Mechanistically, we found that persistent infection with H. pylori reduces the sensitivity of TLR6 to bacterial components and regulates the expression of inflammatory cytokines in GES-1 cells through TLR6/JNK signaling. The TLR6 agonist obviously alleviated inflammation in vitro and in vivo. Promising results suggest that TLR6 may be a potential candidate immunotherapy drug for H. pylori infection.

ABSTRACT Helicobacter pylori is a microaerophilic Gram-negative bacterium that resides in the human stomach and is classified as a class I carcinogen for gastric cancer. Numerous studies have demonstrated that H. pylori infection plays a role in regulating the function of host cells, thereby contributing to the malignant transformation of these cells. However, H. pylori infection is a chronic process, and short-term cellular experiments may not provide a comprehensive understanding of the in vivo situation, especially when considering the lower oxygen levels in the human stomach. In this study, we aimed to investigate the mechanisms underlying gastric cell dysfunction after prolonged exposure to H. pylori under hypoxic conditions. We conducted a co-culture experiment using the gastric cell line GES-1 and H. pylori for 30 generations under intermittent hypoxic conditions. By closely monitoring cell proliferation, migration, invasion, autophagy, and apoptosis, we revealed that sustained H. pylori stimulation under hypoxic conditions significantly influences the function of GES-1 cells. This stimulation induces epithelial-mesenchymal transition and contributes to the propensity for malignant transformation of gastric cells. To confirm the in vitro results, we conducted an experiment involving Mongolian gerbils infected with H. pylori for 85 weeks. All the results strongly suggest that the Nod1 receptor signaling pathway plays a crucial role in H. pylori -related apoptosis and autophagy. In summary, continuous stimulation by H. pylori affects the functioning of gastric cells through the Nod1 receptor signaling pathway, increasing the likelihood of cell carcinogenesis. The presence of hypoxic conditions further exacerbates this process. IMPORTANCE Deciphering the collaborative effects of Helicobacter pylori infection on gastric epithelial cell function is key to unraveling the development mechanisms of gastric cancer. Prior research has solely examined the outcomes of short-term H. pylori stimulation on gastric epithelial cells under aerobic conditions, neglecting the bacterium’s nature as a microaerophilic organism that leads to cancer following prolonged stomach colonization. This study mimics a more genuine in vivo infection scenario by repeatedly exposing gastric epithelial cells to H. pylori under hypoxic conditions for up to 30 generations. The results show that chronic exposure to H. pylori in hypoxia substantially increases cell migration, invasion, and epithelial-mesenchymal transition, while suppressing autophagy and apoptosis. This highlights the significance of hypoxic conditions in intensifying the carcinogenic impact of H. pylori infection. By accurately replicating the in vivo gastric environment, this study enhances our comprehension of H. pylori ’s pathogenic mechanisms in gastric cancer.

Background: The genetic quality of laboratory mice may have a direct impact on the results of research. Therefore, it is essential to improve genetic monitoring methods to guarantee research quality. However, few current methods boast high efficiency, high throughput, low cost, and general applicability at the same time. Methods: First, we got 34 SNP loci from previous studies for inbred strains and screened out 15 loci with good polymorphism for outbred groups from these 34 loci. Then, by using the Luminex xTAG assay, we tested inbred strains and outbred groups. Results: We tested commonly used inbred strains and five DNA samples from the International Council for Laboratory Animal Science, obtaining correct genotyping results. Additionally, some loci were potentially confirmed to be useful for distinguishing C57BL/6 and BALB/c mouse substrains. Furthermore, we tested three outbred groups and analyzed the genetic structure, and we compared the results of the SNP markers by xTAG assay to the STR markers by PCR, the trends of the three groups are the same. Conclusions: In our studies, the panels could meet the requirements for method promotion and provide a good choice for the genetic monitoring of inbred and outbred mice.

Hepatocellular carcinoma (HCC), a prevalent solid carcinoma of significant concern, is an aggressive and often fatal disease with increasing global incidence rates and poor therapeutic outcomes. The etiology and pathological progression of non-alcoholic steatohepatitis (NASH)-related HCC is multifactorial and multistage. However, no single animal model can accurately mimic the full NASH-related HCC pathological progression, posing considerable challenges to transition and mechanistic studies. Herein, a novel conditional inducible wild-type human HRAS overexpressed mouse model (HRAS-HCC) was established, demonstrating 100% morbidity and mortality within approximately one month under normal dietary and lifestyle conditions. Advanced symptoms of HCC such as ascites, thrombus, internal hemorrhage, jaundice, and lung metastasis were successfully replicated in mice. In-depth pathological features of NASH- related HCC were demonstrated by pathological staining, biochemical analyses, and typical marker gene detections. Combined murine anti-PD-1 and sorafenib treatment effectively prolonged mouse survival, further confirming the accuracy and reliability of the model. Based on protein-protein interaction (PPI) network and RNA sequencing analyses, we speculated that overexpression of HRAS may initiate the THBS1-COL4A3 axis to induce NASH with severe fibrosis, with subsequent progression to HCC. Collectively, our study successfully duplicated natural sequential progression in a single murine model over a very short period, providing an accurate and reliable preclinical tool for therapeutic evaluations targeting the NASH to HCC continuum.

目的 对 2021 年能力验证计划肽酶-3 样本的均匀性和稳定性进行研究.方法 根据 CNAS-CL04 和 GB/T 14927.1-2008,对肽酶-3 样本进行均匀性和稳定性检验.考察 4 个温度(37℃,室温,4℃和-20℃)条件下和运输条件下的样本稳定性.结果 肽酶-3 样本,均匀性检验合格.在 37℃条件下可以稳定 6 h;在室温条件下可以稳定 7 d;在 4℃条件下,可以稳定 4 周;-20℃条件下,可以稳定 2 个月;在运输条件下,可以稳定 7 d.结论 肽酶-3 样本的均匀性和稳定性检验结果满足开展能力验证计划的要求.

目的 对北京地区实验动物中猴痘病毒(MPXV)感染情况进行调查.方法 参照世界卫生组织(WHO)MPXV实验室检测指导意见,采用荧光PCR法检测北京地区实验动物中MPXV,共642只动物,包括猴127只、小鼠215只、大鼠80只、豚鼠40只、地鼠10只、兔85只、犬85只.其中猴采集咽拭子,大小鼠、豚鼠和地鼠等采集盲肠,兔和犬采集肛拭子,采集的样本加入适量灭菌PBS,盲肠样本充分匀浆,拭子样本涡旋振荡1 min,-70℃反复冻融3次,离心取上清提取核酸,荧光PCR检测MPXV DNA,最后对结果进行分析.结果 经荧光PCR检测,127只猴、215只小鼠、80只大鼠、40只豚鼠、10只地鼠、85只兔、85只犬MPXV DNA均为阴性.结论 目前未在北京地区实验动物中发现MPXV感染.

目的 建立8种鼠源性病毒的实时荧光定量PCR(real-time fluorescent quantitative PCR,Q-PCR)检测方法,并进行验证.方法 通过4个实验室验证Q-PCR法的特异性、灵敏度及精密性,同时进行病毒模拟污染试验及盲样检测,并将检测结果进行比对.采用Q-PCR法检测26批SARS-CoV-2疫苗生产用单抗细胞株及15批其他鼠源性制品.结果 用于8种鼠源病毒检测的Q-PCR法与同科同属或其他鼠源病毒无交叉反应;除实验室2对鼠痘病毒(又名脱脚病病毒)(ectromelia virus,EctV/Mouse Pox,MPV)检测的灵敏度为2× 102 copies/μL外,实验室2对其他7种病毒及其他3个实验室对8种鼠源性病毒的检测灵敏度均为2 × 101copies/μL;除实验室3对鼠腺病毒(mouse adenovirus,MAdV)检测拷贝数试验间CV为37.58％外,实验室3对其他7种病毒和其他3个实验室对8种病毒检测的试验内、试验间Ct和拷贝数CV均＜25％.病毒模拟污染试验检测灵敏度均符合参数要求;4个实验室盲样检测结果符合率为100％.26批SARS-CoV-2疫苗生产用单抗细胞株及15批其他鼠源性制品8种鼠源病毒检测结果均为阴性.结论 鼠源性病毒的Q-PCR法具有良好的特异性、灵敏度及精密性,可用于鼠源性生物制品的检测.

目的 建立小鼠肺炎病毒RT-PCR检测方法,用于实验动物及实验动物相关样本的检测.方法 选择小鼠肺炎病毒(pneumonia virus of mice,PVM)G基因保守序列设计合成引物,建立RT-PCR方法,并进行方法的特异性、敏感性、重复性和稳定性验证.应用建立的方法对日常送检的27只小鼠、9只大鼠、5只沙鼠肺组织样本和19只PVM感染小鼠肺组织样本,及8份国际实验动物理事会(the International Council for Laboratory Animal Science,ICLAS)国际比对样本进行检测.结果 建立的方法与仙台病毒、呼肠孤病毒Ⅲ型、汉坦病毒、淋巴细胞脉络丛脑膜炎病毒无交叉反应.能够检测PVM DNA最小模板浓度为8.77×102拷贝/μL,可检测病毒最小滴度为10-4.0/mL.用放置-30℃冰箱保存12个月的引物和PVM质粒进行PCR检测,仍能扩增到约249 bp的目的条带.用建立的方法检测日常送检的27只小鼠、9只大鼠、5只沙鼠肺组织样本,结果均为阴性;检测19只PVM感染小鼠肺组织样本,有7只小鼠肺组织样本结果阳性;检测8份ICLAS国际比对样本,有1份样本小鼠肺炎病毒核酸阳性,均符合预期结果.结论 建立的PVM RT-PCR方法特异、敏感,重复性、稳定性好,能够用于小鼠、大鼠、沙鼠等实验动物的监测及其相关样本的检测.

Objective To analyze the status of proficiency testing samples in the field of laboratory animal testing, and to evaluate the role of the testing samples in the laboratory animal laboratory proficiency testing program.MethodsA retrospective study was conducted to compare the 30 proficiency testing plans and reports in the field of laboratory animal organized by National Institute for Food and Drug Control (NIFDC) from 2013 to 2022. The type, quantity, preparation method, homogeneity and stability of the proficiency testing samples, as well as the satisfaction of the participating units to the samples were analyzed. Then, the supply of new samples, the addition of quantitative projects, the improvement of stability research and the attention to customer needs were discussed.ResultsThe 30 proficiency testing programs included 10 items in the direction of bacteria, 10 items in the direction of virus and 10 items in the direction of genetics in the field of experimental animals. A total of 80 non-nucleic acid samples (including serums, feces, tracheal flushing fluid and proteins) and 4 nucleic acid samples were provided, all of which were qualitative samples. Compared with nucleic acid samples, non-nucleic acid samples always required large numbers of animals for sample preparation. The pass rate of homogeneity verification of the sample was 100%, but there is no quantitative study at present. Stability verification can be conducted for the short-term storage, transportation, freeze-thaw, different concentrations and terminal stability of different samples, but it lacks long-term stability verification.ConclusionIn the past ten years, the proficiency testing samples have basically met the basic requirements of the proficiency testing plan of laboratory animal testing area. However, the provision of nucleic acid samples should be reasonably increased; quantitative research samples and projects should be added; the influence of proficiency testing in the field of laboratory animal testing should be continuously expanded and maintain high-quality services.

目的 通过对国际实验动物科学理事会(the International Council for Laboratory Animal Science,ICLAS)能力验证计划(Performance Evaluation Program,PEP)提供的4份病毒盲样进行检测,从而对实验室病毒检测能力进行验证,进一步提高实验室的病毒检测水平.方法 对ICLAS提供的盲样信息进行分析,提取核酸进行PCR检测并对阳性产物进行测序验证.对诺如病毒阴性盲肠内容物进行不同倍数的稀释后,与诺如病毒培养液1:1混合,使用两种不同的核酸提取试剂盒进行核酸提取,通过荧光定量PCR方法进行诺如病毒核酸检测,比较检测的核酸拷贝数.结果 经过检测确定ICLAS提供的盲样分别为小鼠脑脊髓炎病毒、小鼠诺如病毒、小鼠轮状病毒以及小鼠乳酸脱氢酶增高症病毒.比较不同稀释度的盲肠内容物诺如病毒荧光定量PCR检测结果发现,使用病毒DNA/RNA提取试剂盒提取核酸,稀释度不高于1:16时,检测结果相比于病毒对照出现不同程度的偏差,稀释度为1:32时检测结果与病毒对照检测结果相近.使用病毒RNA提取试剂盒提取核酸,稀释度不低于1:8时,检测结果与病毒对照相近.结论 通过国际比对能力验证,说明本实验室病毒检测能力可靠,且经过污染实验研究,粪便样本1:8稀释后使用病毒RNA提取试剂盒提取核酸为粪便中诺如病毒检测的最佳预处理方法.

为了探索纳米孔测序技术在动物传染病病原快速诊断中的应用价值,采集1只发生腹泻实验兔的肝脏和脾脏进行核酸提取、建库和纳米孔测序分析,同时对该腹泻的实验兔分别进行细菌分离鉴定和腹泻相关病毒的血清学检测.结果显示:纳米孔测序方法运行3h后,获得1.9GB的数据量,检出756 521条序列,平均序列长度为2 549.9个碱基.根据对测序数据的生物信息学分析表明,引起实验兔腹泻的病原可能为大肠杆菌.对同批送检的6只实验兔采用常规细菌分离鉴定方法在实验兔的心脏血样品、肝脏、肺脏和回盲部内容物中只分离到了大肠杆菌.对腹泻相关病毒的血清学检测结果显示6只送检的兔均为兔轮状病毒阴性.本研究采用纳米孔测序技术对发生腹泻的实验兔进行诊断,其结果与常规实验室检测方法获得的结果相符合,表明纳米孔技术在动物传染病病原快速检测中具有潜在的应用价值.

目的 分析温度-时间、浓度、冻融、运输条件和不同品系来源对苹果酸酶-1(malic enzyme-1,ME-1)和异柠檬酸脱氢酶-1(isocitrate dehydorgenase-1,IDH-1)标准样品的影响.方法 制备C57BL/6、BALB/c和CBA三种近交系小鼠来源的标准样品,经均匀性测定后,随机抽样按照GB/T 14927.1—2008中的检测方法进行测试.温度-时间因素以4℃、室温(RT)和37℃这3个温度为观测纵坐标,1、2、3、7、14和21 d为观测纵坐标.在每个坐标交点,每品系分别随机抽取两管.浓度因素按照每品系一管进行原倍、1/2、1/4和1/8倍进行梯度测试.冻融因素,每品系3管,-20℃冻存2 h至室温融化3次为条件测试.所有实验均为相同的运输条件、干冰冷链及北京往返沈阳、西安、成都三地测试.结果 不同品系来源标准样品在温度-时间、浓度、冻融和运输条件下无显著差异.各标准样品21 d内在4℃及室温(RT)条件下,均能稳定存在,但37℃仅能稳定存在2 d.原倍稀释至1/8浓度、3次冻融和干冰运输三地往返后,样品检测结果显示稳定.结论 制备的ME-1和IDH-1标准样品具备均一性、稳定性和可比性,满足能力验证计划需求.