Rapid genotyping of human rotavirus using SYBR green real-time reverse transcription-polymerase chain reaction with melting curve analysis

AIM: To develop a real-time reverse transcriptionpolymerase chain reaction(RT-PCR) assay to genotype rotavirus(G and P) in Alberta from January 2012 to June 2013. METHODS: We developed and validated a different approach to perform rotavirus G and P genotyping using a two-step SYBR green RT-PCR(rt-g PCR) by selecting genotype-specific primers of published conventional RT nested PCR(cn RT-PCR) assay and optimizing the amplification conditions. c DNA was first synthesized from total RNA with Super Script? Ⅱ reverse transcriptase kit followed by amplication step using monoplex SYBR green real-time PCR. After the PCR reaction, melting curve analysis was used to determine specific genotype. Sixteen samples previously genotyped using cn RT-PCR were tested using the new assay and the genotyping results were compared as sensitivity analysis. Assay specificity was evaluated by testing other gastroenteritis viruses with the new assay. The amplicon size of each available genotype was determined by gelelectrophoresis and DNA sequences were obtained using Sanger-sequencing method. After validation and optimization, the new assay was used to genotype 122 pediatric clinical stool samples previously tested positive for rotavirus using electron microscopy between January2012 and June 2013.RESULTS: The new rt-g PCR assay was validated and optimized. The assay detected G1 to G4, G9, G12 and P[4] and P[8] that were available as positive controls in our laboratory. A single and clear peak of melting curve was generated for each of specific G and P genotypes with a Tm ranging from 80 ℃ to 82 ℃. The sensitivity of rt-g PCR was comparable to cn RT-PCR with 100% correlation of the 16 samples with known G and P genotypes. No cross reaction was found with other gastroenteritis viruses. Using the new rt-g PCR assay, genotypes were obtained for 121 of the 122 pediatric clinical samples tested positive for rotavirus: G1P[8](42.6%), G2P[4](4.9%), G3P[8](10.7%), G9P[8](10.7%), G9P[4](6.6%), G12P[8](23.0%), and unknown GP[8](0.8%). For the first time, G12 rotavirus strains were found in Alberta and G12 was the second most common genotype during the study period. Gel electrophoresis of all the genotypes showed expected amplicon size for each genotype. The sequence data of the two G12 samples along with other genotypes were blasted in NCBI BLAST or analyzed with Rota C Genotyping tool(http://rotac.regatools.be/). All genotyping results were confirmed to be correct.CONCLUSION: rt-g PCR is a useful tool for the genotyping and characterization of rotavirus. Monitoring of rotavirus genotypes is important for the identification of emerging strains and ongoing evaluation of rotavirus vaccination programs.

Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription

Quantitative real-time reverse transcription-polymerase chain reaction （qPCR） is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxJn selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference 13-111 tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

Detection of circulating hepatocellular carcinoma cells in peripheral venous blood by reverse transcription-polymerase chain reaction

Objective: To detect circulating hepatocellular carcino-ma by demonstrating hepatocellular carcinoma cells orhepatocyte-associated mRNA in the nuclear cell com-ponent of peripheral blood (PBL).Methods: Peripheral blood (5 ml) samples were ob-tained from 93 patients with hepatocellular carcinoma(HCC) and from 33 control subjects (9 with liver cir-rhosis after hepatitis B,14 with chronic hepatitis B,10with normal liver function). To identify HCC cells inperipheral blood, liver-specific human alpha-fetopro-tein (AFP) mRNA was amplified from total RNA ex-tracted from whole blood by reverse transcription-polymerase chain reaction.Results: AFPmRNA was detected in 50 blood samplesfrom the HCC patients (50/93, 53.8%). In contrast,there were no clinical control patients whose samplesshowed detectable AFPmRNA in PBL. The presence ofAFPmRNA in blood seemed to be correlated with thestage (by TNM classification) of HCC, the serum AFPvalue, and the presence of intrahepatic metastasis,portal vein thrombosis, tumor diameter and/or distantmetastasis. In addition, AFPmRNA was detected in theblood of 21 patients with metastasis at extrahepaticorgans (100%) in contrast to 29 (40.3%)of 72 pa-tients without metastasis.Conclusion: The presence of AFPmRNA in peripheralblood may be an indicator of malignant hepatocytes,which might predict hematogenous spreading metasta-sis of tumor cells in patients with HCC.

Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

A quantitative real time reverse-transcription polymerase chain reaction （qRT-PCR） assay with specific primers recommended by the World Health Organization （WHO） has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin （HA） genes of the pandemic HlN1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic HIN1/2009 virus infection.

Positive reverse transcription-polymerase chain reaction assay results in patients recovered from COVID-19: Report of two cases

BACKGROUND Coronavirus disease 2019(COVID-19)has spread around the globe.On February 28,2020,the World Health Organization adjusted the risk of spread and impact of COVID-19 to“very high”at the global level.Studies have mainly focused on the etiology,epidemiology,and treatment of COVID-19 to limit further spread and the negative impact of the disease,while less attention has been devoted to the follow-up and reexamination of patients who recovered from COVID-19 or were released from quarantine.CASE SUMMARY This study reports two cases where patients who had negative reverse transcription-polymerase chain reaction(RT-PCR)test results and met the criteria for discharge subsequently had positive RT-PCR test results.The clinical manifestations and computed tomography(CT)findings of these patients were examined.The conversion of RT-PCR test results in these two patients may be related to false-negative and false-positive outcomes of the test.CT images helped track improvement of pulmonary lesions.CONCLUSION The timing of discharge of COVID-19 patients should be determined by comprehensive analysis of CT images and RT-PCR test results.

Development and application of a real-time polymerase chain reaction method for Campylobacter jejuni detection

AIM:To develop a real-time polymerase chain reaction(PCR) method to detect and quantify Campylobacter jejuni(C.jejuni) from stool specimens.METHODS:Primers and a probe for real-time PCR were designed based on the specific DNA sequence of the hipO gene in C.jejuni.The specificity of the primers and probe were tested against a set of Campylobacter spp.and other enteric pathogens.The optimal PCR conditions were determined by testing a series of conditions with standard a C.jejuni template.The detection limits were obtained using purified DNA from bacterial culture and extracted DNA from the stool specimen.Two hundred and forty-two specimens were analyzed for the presence of C.jejuni by direct bacterial culture and real-time PCR.RESULTS:The optimal PCR system was determined using reference DNA templates,1 × uracil-DNA glycosylase,3.5 mmol/L MgCl 2,1.25 U platinum Taq polymerase,0.4 mmol/L PCR nucleotide mix,0.48 μmol/L of each primer,0.2 μmol/L of probe and 2 μL of DNA template in a final volume of 25 μL.The PCR reaction was carried as follows:95 ℃ for 4 min,followed by 45 cycles of 10 s at 95 ℃ and 30 s at 59 ℃.The detection limit was 4.3 CFU/mL using purified DNA from bacterial culture and 10 3 CFU/g using DNA from stool specimens.Twenty(8.3%,20/242) C.jejuni strains were isolated from bacterial culture,while 41(16.9%,41/242) samples were found to be positive by realtime PCR.DNA sequencing of the PCR product indicated the presence of C.jejuni in the specimen.One mixed infection of C.jejuni and Salmonella was detected in one specimen and the PCR test for this specimen was positive.CONCLUSION:The sensitivity of detection of C.jejuni from stool specimens was much higher using this PCR assay than using the direct culture method.

Expression of cellular fibronectin mRNA in adult periodontitis and peri-implantitis: a real-time polymerase chain reaction study

Cellular fibronectin （cFn） is a type of bioactive non-collagen glycoprotein regarded as the main substance used to maintain periodontal attachment. The content of cFn in some specific sites can reflect the progress of periodontitis or peri-implantitis. This study aims to evaluate the expression of cFn messenger RNA （mRNA） in tissues of adult periodontitis and peri-implantitis by real-time fluorescent quantitative polymerase chain reaction （PCR） and to determine its clinical significance. A total of 30 patients were divided into three groups of 10： healthy, adult periodontitis and peri-implantitis. Periodontal tissue biopsies （1 mmx I mmx I mm） from each patient were frozen in liquid nitrogen. Total RNA was extracted from these tissues, and the content, purity and integrity were detected. Specific primers were designed according to the sequence, and the mRNA expression levels of cellular fibronectin were detected by real-time PCR. The purity and integrity of the extracted total RNA were both high, and the specificity of amplified genes was very high with no other pollution. The mRNA expression of cFn in the adult periodontitis group （1.526＋0.441） was lower than that in the healthy group （3.253＋0.736）. However, the mRNA expression of cFn in the peri-implantitis group （3.965＋0.537） was significantly higher than that in the healthy group. The difference revealed that although both processes were destructive inflammatory reactions in the periodontium, the pathomechanisms were different and the variation started from the transcription level of the cFn gene.

Real-time polymerase chain reaction for the diagnosis of necrotizing herpes stromal keratitis

AIM： To design, optimize and validate a rapid,internally controlled real-time polymerase chain reaction（RT-PCR） test for herpes simplex virus（HSV） in the diagnosis of necrotizing herpes stromal keratitis.· METHODS： Tears alone or together with corneal epithelium scrapings from 30 patients（30 eyes）suspected of necrotizing herpes stromal keratitis were tested for HSV DNA by RT-PCR. The samples were collected during the first visit and then on the subsequent 7, 14, 28, 42, and 56 d. The symptoms of the patients were scored before treatment to determine the correlation between HSV concentration in the corneal epithelium scrapings and clinical scores.·RESULTS： The positive rate（46.4%） in the corneal epithelium group before the therapy was significantly higher than that（13.3%） in the tears group（P =0.006）.There were 13 positive HSV patients before the therapy,the concentration of HSV DNA in corneal epithelium scrapings group was significantly higher than that in the tears group（paired t-test, P =0.0397）. Multilevel mixedeffects model analysis showed that the difference between the corneal epithelium scrapings group and the tears group was statistically significant（P =0.0049）. The Spearman rank correlation analysis indicated a positive correlation between the HSV concentration in the corneal epithelium scrapings and clinical scores before the treatment（r =0.844, P〈 0.0001）.· CONCLUSION： RT-PCR appears to be a powerful molecular tool for the diagnosis of necrotizing herpes stromal keratitis.

4种植物源性成分多重real-time PCR检测方法的建立及其在食用淀粉中的应用

建立一种可同时快速检测红薯、木薯、马铃薯、玉米源性成分的多重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法。分别以红薯g3pdh基因、木薯g3pdh基因、马铃薯UGPase基因、玉米zSSIIb基因为靶基因设计特异性引物和TaqMan探针,以18S rRNA基因为内参基因,建立多重real-time PCR方法,开展方法学验证,并对不同掺入比例模拟样品和实际淀粉样品进行检测。结果显示,该方法具有高通量、特异性强、灵敏度高等优点。与15种非目标源性均无交叉反应;对目标DNA的检测灵敏度可达到3×10^(-3) ng/μL,且具有良好的线性关系和扩增效率;对淀粉样品的检出限可达0.1%,对50份实际样品进行检测,结果与参比方法一致,说明建立的多重real-time PCR法可用于食用淀粉种类掺假鉴别检测。

Detection of hepatocellular carcinoma cells in the peripheral blood with reverse--transcription polymerase chain reaction

In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samples of 113 cases of HCC and 69 controls (including 30 cases of liver cirrhosis, 9 cases of metastatic liver cancer and 30 normal subjects). 20/43 (46. 5% ) cases of HCC and 2/30 (6. 7% ) cases of liver cirrhosis are positive and the cases of nletastatic liver cancer and normal controls were negative for human AFP(hAFP) rnRNA. The presence of hAFP mRNA in the peripheral blood seems to be correlated with intrahepatic and distant nletastasls of HCC and portal vein thrombosis. It is concluded that the presence of hAFP mRNA in the peripheral hloocl is an indicator of circulating HCC cells and can be used to diagnose the rnetastasisof HCC through henlatogenous route and RT-PCR amplification of hAFP mRNA is a sensitive and specificprocedure for detecting circulating cells of HCC.

马乳酒样乳杆菌马乳酒样亚种real-time PCR检测方法的建立与应用

本研究建立了一种特异性实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)检测方法,根据模式菌株马乳酒样乳杆菌马乳酒样亚种ZW3的16S rDNA序列和全基因组序列设计筛选特异性引物,采用SYBR Green I荧光染料建立real-time PCR方法,并对方法的特异性、灵敏度、重复性和混合体系等进行检测。结果表明,本研究所建立的方法特异性强、灵敏度高、重复性好,建立real-time PCR的标准曲线,其决定系数R2为0.965,具有良好的线性关系,且在马乳酒样乳杆菌马乳酒样亚种及混合体系中可以特异性检出。综上,本研究建立的real-time PCR法可以快速、准确地检测马乳酒样乳杆菌马乳酒样亚种,为马乳酒样乳杆菌的特异性定性定量检测提供了一种新的方法。

垂体后叶粉中3种动物源性成分多重real-time PCR检测方法的建立及应用

目的基于三重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)技术同时快速检测垂体后叶粉中的猪、牛、羊性动物源性成分。方法采用CTAB法提取样品及对照品DNA,以线粒体基因为靶基因设计并合成三重real-time PCR的引物探针,并对建立方法进行验证,用于检测垂体后叶粉样品是否与标识一致。结果猪、牛、羊源性成分的最低检测浓度为10 pg/μL,采用建立的方法检测3批垂体后叶粉样品,均只检测出猪源性成分。结论建立的方法可用于同时检测垂体后叶粉中的猪、牛、羊源性成分,可为相关工作提供技术支持。

一种5重real-time PCR筛查转基因水稻方法的建立

以转基因水稻中最常用的CaMV35S启动子、NOS终止子、Cry1Ab/Ac基因、HPT基因及SPS水稻内标基因为研究对象,利用5种不同的荧光信号(FAM、HEX、Taxas Red、Cy5、Cy5.5)进行多重实时聚合酶链式反应(realtime polymerase chain reaction,real-time PCR)检测方法的研究。通过引物组合筛选、反应体系优化、特异性测试、灵敏度测试、适用性测试等一系列实验,建立了5重real-time PCR方法,灵敏度可达0.032%。此方法具有灵敏度高、结果准确、通量大等优点,可实现水稻中转基因成分的快速、高效检测。

3种致病菌多重real-time PCR检测方法的建立及其在散装即食肉制品中的应用

建立一种同时快速检测沙门氏菌、金黄色葡萄球菌和蜡样芽孢杆菌的三重实时聚合酶链式反应(realtime polymerase chain reaction,real-time PCR)方法。以沙门氏菌invA基因、金黄色葡萄球菌Sa442基因和蜡样芽孢杆菌Cereolysin AB基因为靶基因设计引物和TaqMan探针,建立多重real-time PCR方法,对该方法进行方法学验证,并对实际的散装即食肉制品样品进行检测。结果表明,该方法特异性强、灵敏度高、重复性好。对15株非目标菌进行检测,结果均为阴性;3种致病菌的定量线性浓度范围为10^(2)~10^(8) CFU/mL,且定量检测批内和批间的变异系数均小于2%;沙门氏菌、金黄色葡萄球菌和蜡样芽孢杆菌在未增菌的即食肉制品样品中检出限分别为3.8×10^(2)、4.9×10^(2) CFU/mL和5.7×10^(2) CFU/mL,经增菌5 h后,检出限提高到3.8、4.9 CFU/mL和5.7 CFU/mL。对100份实际样品进行检测,结果与标准方法一致,说明建立的多重real-time PCR法可以用于散装即食肉制品中3种致病菌的检测。

多重real-time PCR技术快速鉴别特种乳中的乳源动物成分

建立一种特种乳中乳源动物成分快速鉴别多重实时聚合酶链式反应技术。通过筛选建立8种不同乳源物种检测的多重实时聚合酶链式反应方法,在一个反应体系里可同时检测4种乳源的特异性靶基因,绝对灵敏度达0.1~5 pg/μL,检出限可达0.1%。同时,建立了高效的DNA提取方法,样品裂解后可在12 min左右完成96个样品的DNA提取,大大缩短了样品前处理时间。采用该方法对市售特色乳产品进行调查,结果显示标识不准确的产品比例达36.36%。

4种动物源性成分多重real-time PCR检测方法的建立及其在驴肉制品检测中的应用

建立一种同时快速检测驴、马、猪及鸭源性成分的四重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法。分别以4种源性成分的Nad5、ATpase6、ATP8、cytb基因为靶基因设计特异性引物和TaqMan探针,以18S rRNA基因为内参基因,建立多重real-time PCR方法,并对该方法进行方法学验证,同时对不同掺入比例模拟样品、不同加工工艺模拟样品和实际驴肉样品进行检测。结果显示,该方法具有高通量、特异性强、灵敏度高等优点。当Ct值≤35.0时,方法对16种非目标源性具有良好特异性;灵敏度可检测到质量浓度为2×10^(-4)ng/μL的模板DNA;对生肉的检出限为肉含量的0.001%,对熟肉制品的检出限为肉含量的0.01%;对100份实际样品进行检测,结果与标准方法一致,说明建立的多重real-time PCR法可用于肉及肉制品中常见掺假源性成分的检测。

全基因组测序和real-time PCR法检测食源性沙门氏菌parC、gyrA基因突变特征

以45株食源性沙门氏菌喹诺酮耐药株为对象,采取全基因组测序和实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)法检测parC、gyrA基因突变位点,并对检测方法可靠性、耐药基因突变特征进行评估和分析。首先将4株沙门氏菌进行二代全基因组测序,根据测序数据分析结果,建立了一种real-time PCR法检测gyrA Asp87Tyr、gyrA Asp87Asn、parC Thr57Ser和parC Ser80Ile这4个突变位点。将沙门氏菌进行qnrS、qnrA、qnrB的real-time PCR检测,发现有31株菌未检出qnr基因。以这31株菌为对象,采取real-time PCR法筛查基因突变位点,结果发现parC Thr57Ser和gyrA Asp87Asn型突变最常见。将real-time PCR阳性的10株菌扩增parC、gyrA基因全长并测序,real-time PCR检测和测序结果完全吻合,说明了real-time PCR检测的可靠性。全基因组测序和real-time PCR法相结合的方法用于耐药基因突变筛查,既可以发现新的基因突变,又可以快速筛查大样本的主要突变类型,可作为沙门氏菌耐药性研究的一种可靠手段。

三重real-time PCR检测副溶血弧菌主要毒力基因

针对编码副溶血弧菌的特异性基因和主要毒力基因tlh、tdh、ureR进行引物及探针设计,通过优化反应条件,建立基于Taqman探针快速检测副溶血弧菌毒力基因的三重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法。利用10株副溶血弧菌和22株非副溶血弧菌对设计的引物及探针进行特异性验证,结果表明设计的引物及探针具有很高的特异性;优化后的引物浓度分别为tdh 0.15μmol/L、tlh 0.15μmol/L、ureR 0.80μmol/L,探针浓度分别为HEX 0.50μmol/L、FAM 0.50μmol/L;该方法即使在高浓度的背景细菌存在下,DNA浓度与Ct值均呈良好的线性关系,检出限为1.8×10^(2)拷贝/mL;以完整菌细胞的悬液为模板时,预变性时间为30 min,扩增检测效果与以基因组DNA(gDNA)为模板相当(ΔCt<1)。本研究建立的三重real-time PCR方法能实现快速定量检测副溶血弧菌,并能有效区分致病性及非致病性副溶血弧菌,为副溶血弧菌的快速定量检测和风险评估提供快速、灵敏、准确的方法。

高灵敏度real-time PCR在低病毒载量病毒性肝病中的临床应用价值

目的 评估高灵敏度实时荧光定量聚合酶链反应(real-time PCR)在病毒性肝病病毒核酸定量检测中的临床应用价值,为该方法的普及应用提供依据.方法 收集2016年1月~2018年12月于湖北省中医院(以下简称“我院”)就诊的病毒性肝病患者895例,同期选取我院100名健康体检者作为对照组.分别应用常规方法和高灵敏度real-time PCR方法检测血清病毒核酸载量,比较两种方法检测结果的差异.对其中低病毒载量患者的生物化学指标、免疫学指标、凝血国际标准化比值(INR)和肝纤维化FIB-4指数的变化趋势、特点及其与病毒核酸载量的相关性进行统计分析.结果 受检人群中低病毒载量患者呈高比例分布.高灵敏度real-time PCR方法检测HBV DNA的阳性率远高于常规方法(P<0.05),而对于HCV RNA,两种方法检测结果的一致性较好.低病毒载量患者的多项生物化学指标波动并不明显,且多项生物化学指标与病毒核酸载量无相关性(P>0.05).大部分低病毒载量乙肝患者表现为HBeAg阴性的低水平复制状态.免疫学诊断指标与病毒核酸载量间存在不同程度的不一致和弱关联度.低病毒载量患者的凝血INR和肝纤维化FIB-4指数均高于对照组,差异有统计学意义(P<0.05),但其与病毒核酸载量无相关性(P>0.05).结论 高灵敏度real-time PCR方法对病毒核酸载量的检测具有不可比拟的优势,结合其他的诊断指标,其可在低拷贝病毒性肝病的病程管理中发挥不可替代的重要作用.

Real-time PCR法检测水产品中异鳞蛇鲭、棘鳞蛇鲭源性成分

目的:建立异鳞蛇鲭、棘鳞蛇鲭实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)检测方法。方法:对比异鳞蛇鲭、棘鳞蛇鲭、大西洋鳕等18种鳕鱼及易掺假品种的线粒体细胞色素C氧化酶I(cytochrome C oxidase subunit I,COI)基因,设计异鳞蛇鲭、棘鳞蛇鲭物种特异性引物/探针,建立两种鱼的real-time PCR检测方法,评估其特异性、绝对灵敏度和相对灵敏度;用该方法对50份市售标称“鳕鱼”制品进行检测,评估其实际使用效果。结果:两种检测方法特异性强,绝对灵敏度为0.002 ng/μL(异鳞蛇鲭)和0.0002 ng/μL(棘鳞蛇鲭),相对灵敏度均达0.01%,满足实际检测需求。在50份市售样品中检出1份含异鳞蛇鲭成分,1份含棘鳞蛇鲭成分,与测序比对结果一致。结论:本研究建立的异鳞蛇鲭和棘鳞蛇鲭源性成分的检测方法特异性强、灵敏度高,为保护消费者权益和水产品掺杂使假的检测提供技术支持。