Development of a Certified Reference Material from Caffeine Solution for Assuring the Quality of Food and Drug Measurements

Caffeine intake by pregnant women, adults and children can be harmful to the health of all particularly fetuses if the intake exceeds the permissible limits. Therefore, it is of fundamental importance to measure its concentration accurately using certified reference materials (CRMs). In the literature, no scientific details are published about the certification of caffeine standard solutions, and therefore, the present article covers this gap. A batch of caffeine solution was prepared in concentration of 1000 mg/kg and bottled. Homogeneity and stability of the candidate reference material were assessed by HPLC-UV and the results showed that the material is homogenous and stable enough. Characterization of the caffeine reference material was performed by HPLC-UV, LC-MS/MS and UV-VIS-NIR spectrophotometer in three different days and the characterization uncertainty was estimated in accordance with the requirements of ISO GUM. The certified value (999.86 ± 8.57 mg/kg) was derived as a weighted mean from the gravimetry and the three characterization methods and the certified uncertainty was calculated according to ISO Guide 35. The produced CRM is of strong interest to the food and drug analytical laboratories for the validity and credibility of their caffeine measurement results.

Certification of a Multicomponent Reference Material from Natural Gas Mixture by Gravimetry and Dual GC-FID/TCD System

Natural gas (NG) is one of the most important sources of energy for industrial and domestic consumption in the present era because it is cheap and free from sulfur impurities. Therefore, accurate and precise measurement of its composition is of fundamental importance for trade reasons. To improve the quality of NG gas measurements, certified reference materials (CRMs) should be used for calibration of measuring equipment in order to ensure the traceability of the measurement results to the SI units. For the traceability purpose, a multicomponent natural gas mixture was prepared gravimetrically as a reference material according to ISO 6142 from pure helium, hydrogen, n-pentane, i-pentane, n-butane, i-butane, propane, ethane, hexane, methane and nitrogen. The preparation was done in two dilution steps in 5 L aluminum cylinders. The calculated mole fractions and associated uncertainties of natural gas components were verified by a dual GC-FID/TCD system in accordance with ISO 6143 calibrated by a series of primary gas mixtures (CRMs) produced by an NMI. The results obtained by gravimetry and by GC measurements have been checked for compatibility as required by ISO 6142 and were found in very good agreement. Details of the preparation and calculation of the mole fractions and uncertainties of all gas components are explained in this article.

Development of a Certified Reference Material from Caffeine Solution for Assuring the Quality of Food and Drug Measurements

Caffeine intake by pregnant women, adults and children can be harmful to the health of all particularly fetuses if the intake exceeds the permissible limits. Therefore, it is of fundamental importance to measure its concentration accurately using certified reference materials (CRMs). In the literature, no scientific details are published about the certification of caffeine standard solutions, and therefore, the present article covers this gap. A batch of caffeine solution was prepared in concentration of 1000 mg/kg and bottled. Homogeneity and stability of the candidate reference material were assessed by HPLC-UV and the results showed that the material is homogenous and stable enough. Characterization of the caffeine reference material was performed by HPLC-UV, LC-MS/MS and UV-VIS-NIR spectrophotometer in three different days and the characterization uncertainty was estimated in accordance with the requirements of ISO GUM. The certified value (999.86 ± 8.57 mg/kg) was derived as a weighted mean from the gravimetry and the three characterization methods and the certified uncertainty was calculated according to ISO Guide 35. The produced CRM is of strong interest to the food and drug analytical laboratories for the validity and credibility of their caffeine measurement results.

Deciphering the population structure and genetic basis of growth traits from whole-genome resequencing of the leopard coral grouper(Plectropomus leopardus)

The leopard coral grouper(Plectropomus leopardus)is a species of significant economic importance.Although artificial cultivation of P.leopardus has thrived in recent decades,the advancement of selective breeding has been hindered by the lack of comprehensive population genomic data.In this study,we identified over 8.73 million single nucleotide polymorphisms(SNPs)through whole-genome resequencing of 326 individuals spanning six distinct groups.Furthermore,we categorized 226 individuals with high-coverage sequencing depth(≥14×)into eight clusters based on their genetic profiles and phylogenetic relationships.Notably,four of these clusters exhibited pronounced genetic differentiation compared with the other populations.To identify potentially advantageous loci for P.leopardus,we examined genomic regions exhibiting selective sweeps by analyzing the nucleotide diversity(θπ)and fixation index(FST)in these four clusters.Using these high-coverage resequencing data,we successfully constructed the first haplotype reference panel specific to P.leopardus.This achievement holds promise for enabling high-quality,cost-effectiveimputationmethods.Additionally,we combined low-coverage sequencing data with imputation techniques for a genome-wide association study,aiming to identify candidate SNP loci and genes associated with growth traits.A significant concentration of these genes was observed on chromosome 17,which is primarily involved in skeletal muscle and embryonic development and cell proliferation.Notably,our detailed investigation of growth-related SNPs across the eight clusters revealed that cluster 5 harbored the most promising candidate SNPs,showing potential for genetic selective breeding efforts.These findings provide a robust toolkit and valuable insights into the management of germplasm resources and genome-driven breeding initiatives targeting P.leopardus.

Equalization Reconstruction Algorithm Based on Reference Signal Frequency Domain Block Joint for DTMB-Based Passive Radar

Channel equalization plays a pivotal role within the reconstruction phase of passive radar reference signals.In the context of reconstructing digital terrestrial multimedia broadcasting(DTMB)signals for low-slow-small(LSS)target detection,a novel frequency domain block joint equalization algorithm is presented in this article.From the DTMB signal frame structure and channel multipath transmission characteristics,this article adopts a unconventional approach where the delay and frame structure of each DTMB signal frame are reconfigured to create a circular convolution block,facilitating concurrent fast Fourier transform(FFT)calculations.Following equalization,an inverse fast Fourier transform(IFFT)-based joint output and subsequent data reordering are executed to finalize the equalization process for the DTMB signal.Simulation and measured data confirm that this algorithm outperforms conventional techniques by reducing signal errors rate and enhancing real-time processing.In passive radar LSS detection,it effectively suppresses multipath and noise through frequency domain equalization,reducing false alarms and improving the capabilities of weak target detection.

Comparison study on measurement of rail weld joint between inertial reference method and multi-point chord reference method

Purpose – Straightness measurement of rail weld joint is of essential importance to railway maintenance. Dueto the lack of efficient measurement equipment, there has been limited in-depth research on rail weld joint with a5-m wavelength range, leaving a significant knowledge gap in this field.Design/methodology/approach – In this study, the authors used the well-established inertial referencemethod (IR-method), and the state-of-the-art multi-point chord reference method (MCR-method). Two methodshave been applied in different types of rail straightness measurement trollies, respectively. These instrumentswere tested in a high-speed rail section within a certain region of China. The test results were ultimatelyvalidated through using traditional straightedge and feeler gauge methods as reference data to evaluate the railweld joint straightness within the 5-m wavelength range.Findings – The research reveals that IR-method and MCR-method produce reasonably similar measurementresults for wavelengths below 1 m. However, MCR-method outperforms IR-method in terms of accuracy forwavelengths exceeding 3 m. Furthermore, it was observed that IR-method, while operating at a slower speed,carries the risk of derailing and is incapable of detecting rail weld joints and low joints within the track.Originality/value – The research compare two methods’ measurement effects in a longer wavelength rangeand demonstrate the superiority of MCR-method.

A Reference Vector-Assisted Many-Objective Optimization Algorithm with Adaptive Niche Dominance Relation

It is still a huge challenge for traditional Pareto-dominatedmany-objective optimization algorithms to solve manyobjective optimization problems because these algorithms hardly maintain the balance between convergence and diversity and can only find a group of solutions focused on a small area on the Pareto front,resulting in poor performance of those algorithms.For this reason,we propose a reference vector-assisted algorithmwith an adaptive niche dominance relation,for short MaOEA-AR.The new dominance relation forms a niche based on the angle between candidate solutions.By comparing these solutions,the solutionwith the best convergence is found to be the non-dominated solution to improve the selection pressure.In reproduction,a mutation strategy of k-bit crossover and hybrid mutation is used to generate high-quality offspring.On 23 test problems with up to 15-objective,we compared the proposed algorithm with five state-of-the-art algorithms.The experimental results verified that the proposed algorithm is competitive.

Review of Two Years of Surgical Activities of the General Surgery Department of the Reference Health Center of Commune I of Bamako Mali

The objectives of this work were to evaluate the surgical activities carried out in the general surgery department of the Reference Health Center of Commune I of Bamako, to describe the sociodemographic characteristics of the operated patients, to determine the main pathologies encountered and to evaluate qualitatively the result of the treatment. In order to improve performance, and the quality of care, and to identify common pathologies in the surgical department, we undertook a retrospective study on surgical activities from January 2009 to December 2010. At the end of this study, out of 474 men and 187 women (equal sex ratio 2.53);we were able to determine the frequency of surgical pathologies. Farmers, housewives and pupils/students were the most represented with 25.9% respectively;20% and 13.3%. The most frequently observed pathologies were wall hernia (44.8%), prostate adenoma (12%) and acute appendicitis (10.5%). The average length of hospitalization was 3.43 days. Infectious complications affected 25 patients (3.8% of cases) and a death rate of 0.45% (i.e. 3 patients). The average cost of care was 53,500 FCFA. Indeed, the reality of surgical practice in health centers was not the same because of the level of skills of practicing surgeons.

一个传奇的品牌,一个经典的系列 KEF与TheReference系列的故事

回顾2023年,KEF的产品又再斩获众多不同定位、不同类型的各种Hi-Fi音响奖项。在更新MAT技术后,KEF全线新Meta系列在音质方面的提升是备受肯定的。其中最受瞩目的自然是KEF当家的Reference系列了。Reference (参考)系列作为KEF产品线中一个非常重要的高端系列,历经多次变革,至今依然是KEF产品线当中最受发烧友欢迎的系列。它不仅是嫡传KEF每个时期应用于旗舰级先进技术的产品,更是KEF最新技术的普及应用和向下移植的承载者。这个刚刚迈过第50个年头的经典系列在这么多年来能够持续获得发烧友和媒体的喜爱实属不易,这个系列是如何一步一步成为经典的?我们来回顾一下KEF这个品牌与参考系列的发展。

Radiation Dose from CT-Scan of Childhood’s Head: Results of the First Ivorian Survey in a Single Study Site

Objectives: This study aims to evaluate the level of X-ray doses used in childhood’s head as Local Diagnostic Reference Levels (LDRLs) in computed tomography (CT) at a university hospital in Côte d’Ivoire. The Diagnostic Reference Level (DRL) have been set up and used to prevent unusually high radiation doses used in radiology departments and is therefore an optimization tool for practices and procedures in medical X-ray imaging for the radiation protection of patients. Methods: A prospective study of volume CT dose index (CTDIvol) and dose length product (DLP) was performed on images of childhood’s head obtained from a CT-scanner of 64 bars equipped with the tube current modulation capability and manufactured by Hitachi Medical System. 122 CT-scan data from 55 childhood’s head were analyzed. The scan data were stratified in four age groups: Results: The 75th percentile of CTDIvol and DLP (set as LDRL) obtained with respect to the stratified age groups are: 22.5 mGy and 452.5 mGy∙cm, 27.7 mGy and 690.6 mGy∙cm, 28 mGy and 722.4 mGy∙cm, 33.6 mGy and 736.8 mGy∙cm respectively. These outcome values increase with respect to the age of pediatric patients and are comparable to DRLs values obtained internationally. Conclusions: Obtaining good image quality while using low dose in children’s head computed tomography for radiation protection require to setup more surveys in Côte d’Ivoire for regional and national DRL. We proposed through this survey LDRLs in terms of CTDIvol and DLP, comparable to international DRLs values. This survey will be strengthened by additional surveys in order to obtain national DRLs for the radiation protection of the child patient in Côte d’Ivoire.

The China Seismological Reference Model project

The importance of developing high-resolution seismic models to improve understanding of tectonic processes and enhance seismic hazard mitigation programs,along with the rapid expansion of seismic coverage in China,called for a seismological reference model to be established in China.The China Seismological Reference Model(CSRM)project was initiated by the National Natural Science Foundation of China with two primary goals:(1)the CSRM would serve as a primary source for the current state of seismological research in China,and(2)the seismic data and constraints used to construct the CSRM would be used as a backbone open-access cyberinfrastructure for future research in seismology.The CSRM project was also intended to promote data exchange and scientific collaboration in seismology in China.Accordingly,two parallel efforts of the project are being pursued:(1)construction of the CSRM,and(2)development of a CSRM product center.The CSRM is jointly constrained by various types of seismic constraints extracted from the seismic data recorded at 4511 seismic stations in continental China following a top-down approach,with the seismic structures in the shallower part of the Earth constrained first.Construction of the CSRM involves three preparation steps:(1)building datasets of various seismic constraints from the seismic data,(2)developing a method to incorporate the constraints of surface wave observations from regional earthquakes into the inversion of the seismic structure,and(3)constructing high-resolution pre-CSRM seismic models of the velocity structure in the shallow crust and the Pn-velocity structure in the uppermost mantle.In the final process,the CSRM will be constructed by jointly inverting all the seismic constraints using the pre-CSRM models as starting models or a priori structures.The CSRM product center(http://chinageorefmodel.org)archives and distributes three types of products:CSRM models,the Level 1 original seismic data used to extract seismic constraints in the construction of the CSRM,and Level 2 data on the seismic constraints derived from the Level 1 data and the inferred earthquake parameters in the construction of the CSRM.The CSRM product center has archived 141 TB of Level 1 data from 1120 permanent broadband stations in the China Seismic Network Center and 3391 temporary stations from various institutions and data centers around the world,as well as 140 GB of Level 2 data on various seismic constraints and inferred event parameters from the construction of the CSRM.The CSRM is expected to provide significant insights into the composition and tectonic dynamics in continental China and to enhance the capability of various seismic hazard mitigation programs in China from near real-time rapid determination of earthquake parameters to an earthquake early warning system.The CSRM could also provide guidance for focuses in future seismological research and the design of future active and passive seismic experiments in China.Several focuses are suggested for future seismological research in China,along with the building of a national cyberinfrastructure to sustain and expand the operations of the CSRM project.

Proposed age-stratified reference intervals of FSH derived from normozoospermic men

Objective:To demonstrate that serum follicle-stimulating hormone(FSH)in men rises with age,and to explore FSH reference intervals of age-related partitioning.Methods:Men aged 20-50 years(n=1190)underwent semen analysis according to World Health Organization(2010)methods.Serum was frozen prior to measurement of FSH by using the Siemens ADVIA Centaur®XP immunoassay system.FSH central 95%intervals after logarithmic transformation based on age were derived from 1037 normozoospermic men.These were then applied to oligozoospermic and azoospermic men.Men producing azoospermic semen samples were further classified as having non-obstructive azoospermia by clinical diagnostic criteria,including genetic analysis and surgical exploration.Results:Serum FSH in normozoospermic men increased with age(P<0.05),and reference intervals were determined with 10-year brackets:21-30 years[(1.0-8.2)IU/L],31-40 years[(1.4-9.5)IU/L],41-50 years[(1.9-12.0)IU/L].The proportion of oligozoospermic men with normal FSH concentrations was less than the normozoospermic men,which in turn was lower among azoospermic men(both P<0.01).The azoospermic men were further broken down according to the nature of the azoospermia as either obstructive or non-obstructive azoospermia,and 86.4%(38/44)men with non-obstructive azoospermia had elevated serum FSH concentrations whereas only 6.7%(1/15)men with obstructive azoospermia had high FSH levels,and this was significantly different(P<0.01).Conclusions:FSH concentrations increase in men between 20-50 years,and clinical interpretation of serum FSH results in men must be made by using age-based reference intervals.

An SIMS Study on the Fingerprint Characteristics of Minerals with Special Reference to Minerals from the Jinningian Granites in the Yunnan Sector of the Xikang-Yunnan Axis

In this paper such minerals as apatite, zircon and biotite from granites were determined and analyzed bysecondary ion mass spectrometry (SIMS) for their isotopic ages, REE composition, trace elements and volatilecomponents with an attempt to shed light on the origin and formation environments of the Jinningian granitesin the Yunnan sector of the Kangdian (Xikang- Yunnan) earth's axis. The results show that the apatites,zircons and biotites of different geneses possess different fingerprint characteristics. Some correlations havebeen found between trace elements and volatile components contained in the minerals from granties.

Gestation-specific reference intervals for fetal cardiac Doppler indices from 12 to 40 weeks of gestation

We aimed to establish gestation age specific reference intervals for Doppler indices of fetal cardiac function from 12 to 40 weeks of pregnancy. In a cross-sectional observational study of singleton pregnancies, examinations were performed in 221 women evenly distributed across each week of pregnancy. Blood flow through the four cardiac valves was examined with Doppler. For the atrioventricular valves, velocity and duration of early (E) and atrial (A) waves and the interval (a) between E/A complexes was recorded. For the outflow valves, the duration (b), peak and average velocity of flow in systole was measured. Myocardial performance index (MPI) was calculated as (a - b)/b. Outlet valve diameters were measured and cardiac outputs were calculated. Gestation age specific ranges were constructed for all these parameters. We demonstrated that the cardiac output, peak systolic and time-averaged velocity increase with advancing gestation. However the MPI and E/A ratios show little change across gestation. Fetal cardiac physiology can be studied and Doppler indices reliably measured as early as the late first trimester of pregnancy. Establishing gestation age specific ranges for various cardiac indices throughout pregnancy will help the study of development of fetal cardiac function.

RT-qPCR normalization of reference genes in different lifehistory stages of Gracilaria vermiculophylla(Rhodophyta)

The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.

Responses of vegetation yield to precipitation and reference evapotranspiration in a desert steppe in Inner Mongolia,China

Drought,which restricts the sustainable development of agriculture,ecological health,and social economy,is affected by a variety of factors.It is widely accepted that a single variable cannot fully reflect the characteristics of drought events.Studying precipitation,reference evapotranspiration(ET_(0)),and vegetation yield can derive information to help conserve water resources in grassland ecosystems in arid and semi-arid regions.In this study,the interactions of precipitation,ET_(0),and vegetation yield in Darhan Muminggan Joint Banner(DMJB),a desert steppe in Inner Mongolia Autonomous Region,China were explored using two-dimensional(2D)and three-dimensional(3D)joint distribution models.Three types of Copula functions were applied to quantitatively analyze the joint distribution probability of different combinations of precipitation,ET_(0),and vegetation yield.For the precipitation–ET_(0)dry–wet type,the 2D joint distribution probability with precipitation≤245.69 mm/a or ET_(0)≥959.20 mm/a in DMJB was approximately 0.60,while the joint distribution probability with precipitation≤245.69 mm/a and ET_(0)≥959.20 mm/a was approximately 0.20.Correspondingly,the joint return period that at least one of the two events(precipitation was dry or ET_(0)was wet)occurred was 2 a,and the co-occurrence return period that both events(precipitation was dry and ET_(0)was wet)occurred was 5 a.Under this condition,the interval between dry and wet events would be short,the water supply and demand were unbalanced,and the water demand of vegetation would not be met.In addition,when precipitation remained stable and ET_(0)increased,the 3D joint distribution probability that vegetation yield would decrease due to water shortage in the precipitation–ET_(0)dry–wet years could reach up to 0.60–0.70.In future work,irrigation activities and water allocation criteria need to be implemented to increase vegetation yield and the safety of water resources in the desert steppe of Inner Mongolia.

Identification of suitable reference genes for quantitative gene expression analysis in clam Cyclina sinensis under salinity stress and Vibrio infection

The appropriate reference gene is a prerequisite for accurate normalization of gene expression level,and research on suitable reference genes in clam Cyclina sinensis is scarce.To improve the situation,we selected five commonly used housekeeping genes,including β-actin,Elongation factor 1-α(EF1-α),Glyceraldehyde-3-pho sphate dehydrogenase(GAPDH),40S ribosomal protein S18(RPS18),and Tubulin a(TUB-α),then evaluated their expression stability in different adult tissues and under different experimental treatments(salinity stress and Vibrio parahaemolyticus infection).Their expression stability was analyzed by three frequently used programs,geNorm,NormFinder,and BestKeeper.This analysis indicated that multiple genes should be used for normalization,and we concluded that the reference gene combination GAPDH-RPS18-β-actin,should be used for qRT-PCR analysis in different tissues of C.sinensis under normal physiological conditions.For the clams under salinity stress and Vibrio infection,EF1-α-GAPDHRPS18 was recommended as the gene combination for qRT-PCR normalization.TUB-αwas generally poorly ranked by all programs,and should not be used in future studies.This study should provide fundamental support for accurate quantitative gene expression analysis of this species.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Manila Clam Ruditapes philippinarum Under Hypoxic Stress

Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.

Identification of optimal reference genes in golden Syrian hamster with ethanol-and palmitoleic acid-induced acute pancreatitis using quantitative real-time polymerase chain reaction

Background:Acute pancreatitis(AP)is a severe disorder that leads to high morbidity and mortality.Appropriate reference genes are important for gene analysis in AP.This study sought to study the expression stability of several reference genes in the golden Syrian hamster,a model of AP.Methods:AP was induced in golden Syrian hamster by intraperitoneal injection of ethanol(1.35 g/kg)and palmitoleic acid(2 mg/kg).The expression of candidate genes,including Actb,Gapdh,Eef2,Ywhaz,Rps18,Hprt1,Tubb,Rpl13a,Nono,and B2m,in hamster pancreas at different time points(1,3,6,9,and 24 h)posttreatment was analyzed using quantitative polymerase chain reaction.The expression stability of these genes was calculated using Best Keeper,Comprehensive Delta CT,Norm Finder,and ge Norm algorithms and Ref Finder software.Results:Our results show that the expression of these reference genes fluctuated during AP,of which Ywhaz and Gapdh were the most stable genes,whereas Tubb,Eef2,and Actb were the least stable genes.Furthermore,these genes were used to normalize the expression of TNF-αmessenger ribonucleic acid in inflamed pancreas.Conclusions:In conclusion,Ywhaz and Gapdh were suitable reference genes for gene expression analysis in AP induced in Syrian hamster.

Selection of the appropriate reference genes by quantitative real-time PCR in leopard coral groupers Plectropomus leopardus

Leopard coral groupers(Plectropomus leopardus),commercially bred in South China,are a significant economical fish species.In this study,by means of quantitative real-time PCR(qRT-PCR)technology,we assessed the stability of six common reference genes expression and selected the appropriate reference genes in leopard coral groupers with or without Vibrio harveyi stimulation at different time points.These data produced by qRT-PCR was handled via the geNorm,NormFinder,and BestKeeper software.The results revealed all the examined reference genes had manifest tissue-specific expression in different tissues.Prior to V.harveyi stimulation,RPL13 gene was the appropriate reference gene among eleven tissue types(blood,spleen,hepatopancreas,kidney,stomach,gill,heart,skin,muscle,intestine,brain)in leopard coral groupers.Under V.harveyi stimulation,the most reliable reference genes varied from tissue to tissue and were closely hinged upon different time points.At 6-h post-bacterial injection,the appropriate reference genes in hepatopancreas,spleen,kidney,and gill were Actin,B2M,UBCE,and Actin,respectively.At 9-and 12-h post-bacterial injection,the appropriate reference genes in hepatopancreas,spleen,kidney,and gill were RPL13,Actin,Actin,and Actin,respectively.If one reference gene was preferable,RPL13,Actin,Actin,and Actin could be selected as the reference gene in hepatopancreas,spleen,kidney,and gill of leopard coral groupers after V.harveyi infection,respectively.Expression profiles of two target genes(IL-6 and NK-lysin)were used to further validate reliability of these selected appropriate candidates.This study will lay a solid foundation for the future research on qRTPCR analysis of gene expression in leopard coral groupers.