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Establishment of a real-time fluorescent quantitative pcr Assay for Detection of Genetically Modified Maize Line MON88017

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Agricultural Biotechnology 2017年第1期6卷 15-19,22页

作者： Jun SONG Dong WANG Analysis and Test Center Sichuan Academy of Agriculture SciencesChengdu 610066China

In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms （GMOs） and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent quantitative pcr assay was established for detection of genetically modified maize line MON88017. The established method was evaluated based on the specificity, sensitivity, accuracy and measurement uncertainty. The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017. 1.50% MON88017 sample was detected with 29 replica- tions. The average measured value （ 1. 541% ） was close to the actual value （ 1.50% ） and the relative deviation was 2.70%. The variation coefficient of the measured value was 0.110 g ; the recovery was 100.00% and the measurement uncertainty was 0. 096. The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5% confidence level. Thus, the real-time fluorescent quantitative pcr assay established in this study exhibited high specificity, accuracy and sensitivity, which could provide technical support for the safety supervision of genetically modified organ- isms and products in China.

关键词： Genetically modified maize real-time fluorescent quantitative pcr Specificity Sensitivity Accuracy Measurement uncertainty

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Detection of Lactobacillus acidophilus in Fermented Material by real-time fluorescent quantitative pcr

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Animal Husbandry and Feed Science 2016年第1期8卷 54-57页

作者： Guo Zihao Fang Hua Xia Zhisheng Zhu Xiaoshi Sun Zhongchao Yu Hanli Xia Jiaji Shanghai Yuan Yao Biotechnology Co. Ltd.

The species distinctive pcr primer of Lactobacillus acidophilus （ L. acidophilus） was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template, and L. acidophilus in fer- mented material was conducted the quantitative determination by real-time quantitative pcr （RT-pcr）. Analysis on RT-pcr results shown that contents of L. aci- dophilus in the test sample reached 1.5 billion CFU / g. Test results shown that contents of L. acidophilus in fermented material could be detected accurately by the established RT-pcr method in the test. indicating that the established RT-pcr method could be aookued to the detection of L. acidophilus in fermented material.

关键词： real-time fluorescent quantitative pcr Lactobacillus acidophilus quantitative analysis Fermented material

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Methodological study of detecting TREC with real-time fluorescent quantitative pcr in peripheral blood of patients after hematopoietic stem cell transplantation

Methodological study of detecting TREC with real-time fluore...

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第十届亚太国际骨髓移植会议

作者： YIN Ji-xia WU Da-lin XU Wen-juan SUN Jin Lab Center of Nangfang Hospital First Military Medical University Guangzhou 510515 China

＜正＞Objectives For the purpose of exploring the regeneration function of thymus and associated immunity reconstruction process in patients after HSCT, we intended to develop and optimize the real-time florescence quantitative pcr （FQ-pcr） to quantify the TREC levels. Methods We designed primers and TaqMan-MGB probes with reference sequences of TREC and RAG2 downloaded from GENBANK of NCBI web site., we employed RAG2 as cell number control. Then, the

关键词： real-time fluorescent quantitative pcr T-cell receptor excision circles(TREC) hematopoietic Stem Cell Transplantation

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Developmental Changes of GH Gene in Tan Sheep and Correlation Analysis with Slaughter Traits

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Animal Husbandry and Feed Science 2017年第4期9卷 209-212页

作者： Ma Lina Ma Qing Li Yingkang Wang Zhifen Grassland and Livestock Engineering Technology Research Center Ningxia Academy of Agriculture and Forestry Sciences Ningxia Tan Sheep Breeding Farm

[ Objective] The paper was to study the differential expression of growth hormone （GH） gene in different ages and different tissues of Tan sheep. [ Method] Using real-time fluorescent quantitative pcr （RT-pcr） assay, specific primers were designed according to the GH gene sequence published on Gen- Bank. With the total RNA of Tan sheep tissue as the template, the expression levels of GH gene in pectoral muscle, leg muscle and longissimus dorsi muscle at dif- ferent ages were analyzed. [ Result] In three types of muscle tissues, the expression patterns of GH gene in different tissues at the same month of age were pectoral muscle 〉 leg muscle 〉 longissimus dorsi muscle. The expression levels of GH gene were positively correlated with live weight before slaughter, carcass weight and net meat weight, and had significant positive correlation only with net meat weight （ P 〈 0.05 ）. [ Conclusion ] The content of GH gene in different tissues gradually increased with the increasing months of age, and there were extremely significant differences in expression level of GH gene among different months of age or different sites （P〈0. 01）.

关键词： Tan sheep GH gene real-time fluorescent quantitative pcr Expression pattern Correlation analysis

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Comparative Study on Three Methods of Molecular Biology for Detecting Orf Virus

Comparative Study on Three Methods of Molecular Biology for ...

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第七届中国畜牧科技论坛

作者： Xian Simei Li Pengfei Feng Jiang Liu Ai Yang Yu Liu Zongsheng College of Animal Science Guizhou University Key Laboratory of Animal Disease and Veterinary Public Health of Gui zhou Province

Design LAMP primers(F3&B3,BIP & FIP) for the gene Orf Virus(OrfV)B2L,establish detection methods of LAMP,use outer primers(F3&B3) of LAMP,set up two conventional molecular detection methods-pcr and RTFQ-pcr respectively,compare and study the specificity,sensitivity,detection coincidence rate and time limit of the three detection *** results showed that the three methods only conduct special amplification to nucleic acid of OrfV but not to Goat pox virus(GTPV),foot-and-mouth disease virus(FMDV),Peste des Petits Ruminants virus(PPRV) and extraction of nucleic acids from the skin of healthy goats;LAMP method for detecting of OrfV nucleic acid template has the lowest detection limit of 1.0×10 dilution(5.3 fg),detection time limit of about 90 min,and the method RTFQ-pcr has the lowest detection limit of 1.0×10copies/μL(18.2 fg),detection time limit of about 120 min while the conventional method pcr has the lowest detection limit of 1.0×10dilution(5.3 pg) and the detection time limit of about 150 *** conversion,the sensitivity of LAMP is equivalent to that of RTFQ-pcr and their sensitivities are1000 times higher than that of conventional pcr.5 clinical cases of OrfV infection were detected by three methods with the positive rate of 100%and the coincidence rate of 100%.Comparative study on three methods for detecting OrfV with the same pair of primers was established,and the LAMP method becomes the molecular detection means that is suitable for basic level application and promotion with high sensitivity,strong specificity,high-speed and highefficiency,low equipment requirements.

关键词： Orf Virus(OrfV) LAMP real-time fluorescent quantitative pcr conventional pcr

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Changes of the Transcriptional Levels of Molecules Associated with Endogenous Antigen Processing and Presentation in Porcine Skin-derived Dendritic Cells Infected with PCV2 in vivo

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Agricultural Science & Technology 2012年第5期13卷 1089-1092页

作者：李建东李焕荣聂晓华遇奇崔德凤北京农学院动物科学技术学院北京102206

[Objective] This study aimed to investigate the changes of the transcriptional levels of molecules associated with endogenous antigen processing and presenta- tion in porcine skin-derived dendritic cells infected with PCV2 in vivo. [Method] Healthy 40-day-old Landrace piglets were infected with porcine circovirus type 2 （PCV2） and euthanized on the 34, 7rd, 14th, 21st and 35th d post inoculation （DPI）. The porcine skin-derived dendritic cells （DCs） were collected to analyze the transcrip- tional levels of molecules （LMP7, UBP, MHC-I, calreticulin） associated with endogenous antigen processing and presentation by using real-time fluorescent quantitative pcr （real-time FQ-pcr）. [Result] The results showed that the level of LMP7 mR- NAs was reduced significantly on the 3DPI （P〈0.05）; the level of UBP mRNAs was consistently up-regulated, which increased significantly on the 21DPI and 35DPI （P〈 0.05）; the level of MHC-I mRNAs was significantly down-regulated on the 7DPI （P〈 0.05）; the level of calreticulin mRNAs was up-regulated slightly without significant dif- ference. [Conclusion] PCV2 can inhibit the endogenous antigen processing and presentation ability of porcine skin-derived DCs at early stages of infection.

关键词： Porcine circovirus type 2 Skin-derived dendritic cells Endogenous antigen processing and presentation real-time fluorescent quantitative pcr

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