Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (7): 1458-1468.doi: 10.3864/j.issn.0578-1752.2022.07.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Establishment and Application of PCR Assay for Mycoplasma Contamination in Cell Culture and Live Virus Vaccine

GENG RenHao1,2(),LIU Bo1,WANG Fang1,LUO YuFeng1,QU HongFei1,FAN XueZheng1,QIN YuMing1,DING JiaBo1,XU GuanLong1,SHEN QingChun1(),QIN AiJian2()   

  1. 1China Institute of Veterinary Drug Control, Beijing 100081
    2College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, Jiangsu
  • Received:2021-02-08 Accepted:2021-06-21 Online:2022-04-01 Published:2022-04-18
  • Contact: QingChun SHEN,AiJian QIN E-mail:18705279737@163.com;sqcwlx@sina.com;aijian@yzu.edu.cn

RichHTML

25

PDF

160

Abstract:

【Objective】Mycoplasma contamination is prone to occur in biological research and productions. Aimed at the shortage of time-effectiveness and sensitivity of current Pharmacopoeia detection methods, a simple, rapid, specific and sensitive PCR method for mycoplasma was established. 【Method】All the sequences of mycoplasmas were extracted from the datasets file SILVA_123_ SSURef from SILVA database, which contained aligned small (16S/18S, SSU) ribosomal RNA (rRNA) sequences for all three domains of life (Bacteria, Archaea and Eukarya). 181 16S rRNA unique sequences of Mycoplasma (including 139 classified species and 42 unclassified Mycoplasma) were obtained by deduplication of the sequences. After alignment, the hypervariable regions V6 to V9 were selected as the detection target regions, and the detection primers were designed to establish a universal PCR method for mycoplasma detection. 12 kinds of mycoplasmas or 2 kinds of acholeplasmas were selected to verify the detection range of the PCR method; 6 kinds of passage cells from different animal and 3 kinds of common bacteria were selected for specific verification; 5 kinds of mycoplasmas and a kind of acholeplasma were selected for sensitivity test. In order to evaluate the performance of the assay in clinical application, 17 batches of animal live virus vaccines ( for 6 kinds of animals) and 24 samples of 8 kinds of cell cultures were subjected to test, which were compared with the classical culture method for mycoplasmas. 【Result】A general PCR method for mycoplasma was established. The detection primers were composed of 2 upstream primers (5'-GCAAARCTATRGARAYA TAGYVGAG-3' and 5'- GCAAAGGCTTAGAAATAAGTTCGGAG-3') and a downstream primer (5'- CCARCTCYCATRGTKTGA CGG - 3'), and the mixing ratio of the primers was 3:1:4. The optimal annealing temperature was 56℃. The PCR method was used to detect 14 mycoplasma species, and the results showed that 396 bp-413 bp specific bands had been amplified, which indicated that the detection range of the PCR method satisfied the detection requirements; No specific bands were amplified from 6 kinds of animal cells and 3 kinds of common bacteria with the PCR assay. The results of sensitivity test showed that the detection limit of the PCR method was 20-200 CCU, and the corresponding nucleic acid was 1.5-15.0 pg. The detection results of 17 batches of live virus vaccines and 24 cell samples were almost consistent with those of culture method, which indicated that the PCR method established in this study was consistent with culture method well, and was more sensitive. 【Conclusion】The PCR assay established in this study was specific, sensitive, easy and fast, suitable for rapid detection of mycoplasma contamination in cells and live virus vaccine.

Key words: cell, live virus vaccine, Mycoplasma, 16S rRNA, PCR

Fig. 1

Results of 16S rRNA gene sequence alignment and the binding locations of Mycoplasma PCR primers"

Fig. 2

The result of primer verification for mycoplasma detection PCR 1: DL2000Marker; 2: Positive control; 3: Negative control; 4: M.hyorhinis(CVCC361); 5: M.synoviae(CVCC385); 6: M.Galliscepticum(CVCC353); 7: M.bovis XBY01 strain; 8: M.hyopneumoniae(CVCC4049); 9: M.iowae(CVCC364); 10: M.hominis HB1 strain; 11:M.anatis BSJ52 strain; 12: M. ovis(CVCC380); 13: M.iners(CVCC363); 14: M.arginini(CVCC346); 15: A.laidlawii(CVCC2406); 16: M.orale(CVCC379); 17: A.axanthum 338 strain; 18: E.coli DH5α; 19: Marc145 cell"

Fig. 3

Optimization results of the PCR assay for Mycoplasma detection a to f: The PCR results with the annealing temperature of 53℃, 54℃, 55℃, 56℃, 57℃ and 58℃, respectively. M: DL2000 Marker; 1: M. galliscepticum; 2: M. synoviae; 3: M.hyorhinis; 4: E.coli; 5: Marc145 cell"

Fig. 4

Specificity of the PCR assay for Mycoplasma detection 1: DL2000 Marker; 2: Positive control; 3: Negative control; 4: MDBK cell; 5: CEF cell; 6: Sf9 cell; 7: Marc145 cell; 8: ST cell; 9: SP2/0 cell; 10: E.coli DH5α; 11: Salmonella CVCC2139; 12: C.perfringens CVCC1125"

Fig. 5

Sequencing result of the PCR production of the positive control"

Fig. 6

Sensitivity of the PCR assay for Mycoplasma detection 1,8,15:DL2000 Marker; Figure a, 2-7: M. galliscepticum deluted to 10-1- -6; 9-14: M.hyorhinis deluted to 10-1- -6; 16-21: M. iowae deluted to 10-1- -6 . Figure b, 2-7: M.arginini deluted to 10-1- -6; 9-14: M.orale deluted to 10-1- -6; 16-21: A.laidlawii deluted to 10-1- -6"

Table 1

The results of the veterinary live vaccines Mycoplasma detection with the PCR assay and traditional culture method"

类别
category		 生物制品名称
Biological products		 原材料
Materials		 阳性结果数Number of positive samples
PCR检测
PCR detection		 培养法检测Cultivationa
细胞
Cells		 健康细胞8株（种）
8 strains(8 species) of healthy cell		 / 0/8 0/8
待检细胞16株（8种）
16 strains(8 species) of cell samples		 / 6/16 4/16（6/16）
动物用病毒
活疫苗
Live vaccines for animals		 鸡用病毒活疫苗6批（品种）
6 batches (species) of live vaccines for chicken		 鸡胚、CEF等
Chicken embryos, CEF, etc.		 1/6 0/6
鸭用病毒活疫苗2批（品种）
2 batches (species) of live vaccines for duck		 鸭胚
Duck embryos		 0/2 0/2
鹅用病毒活疫苗1批（品种）
1 batch (species) of live vaccine for geese		 鸭胚
Duck embryos		 0/1 0/1
猪用病毒活疫苗5批（品种）
3 batches (species) of live vaccines for swine		 Vero、PK15、Marc145、ST等
Vero, PK15, Marc145, ST, etc.		 0/5 0/5
犬用病毒活疫苗2批（品种）
2 batches (species) of live vaccines for dog		 鸡胚、Vero、MDCK、PK15等
Chicken embryos, Vero,MDCK, PK15, etc.		 0/2 0/2
草鱼用病毒活疫苗1批（品种）
1 batch (species) of live vaccines for grass carp		 草鱼胚胎细胞
Embryonic cell of grass carp		 0/1 0/1
[1] UPHOFF C C, DREXLER H G. Detection of mycoplasma contaminations. Methods in Molecular Biology (Clifton, N J), 2013, 946:1-13. doi: 10.1007/978-1-62703-128-8_1.
doi: 10.1007/978-1-62703-128-8_1
[2] MEDVEDEVA E S, BARANOVA N B, MOUZYKANTOV A A, GRIGORIEVA T Y, DAVYDOVA M N, TRUSHIN M V, CHERNOVA O A, CHERNOV V M. Adaptation of mycoplasmas to antimicrobial agents: Acholeplasma laidlawii extracellular vesicles mediate the export of ciprofloxacin and a mutant gene related to the antibiotic target. The Scientific World Journal, 2014, 2014:150615. doi: 10.1155/2014/150615.
doi: 10.1155/2014/150615
[3] TEYSSOU R, POUTIERS F, SAILLARD C, GRAU O, LAIGRET F, BOVÉ J M, BÉBÉAR C. Detection of mollicute contamination in cell cultures by 16S rDNA amplification. Molecular and Cellular Probes, 1993, 7(3):209-216. doi: 10.1006/mcpr.1993.1030.
doi: 10.1006/mcpr.1993.1030
[4] ROBINSON L B, WICHELHAUSEN R H, ROIZMAN B. Contamination of human cell cultures by pleuropneumonialike organisms. Science, 1956, 124(3232):1147-1148. doi: 10.1126/science.124.3232.1147.
doi: 10.1126/science.124.3232.1147 pmid: 13380429
[5] VOLOKHOV D V, GRAHAM L J, BRORSON K A, CHIZHIKOV V E. Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques. Molecular and Cellular Probes, 2011, 25(2/3):69-77. doi: 10.1016/j.mcp.2011.01.002.
doi: 10.1016/j.mcp.2011.01.002
[6] 赵翔, 冯建平, 孟淑芳. 支原体检查的核酸检测方法及方法学验证的思考. 中国药事, 2018, 32(8):1020-1027. doi: 10.16153/j.1002-7777.2018.08.003.
doi: 10.16153/j.1002-7777.2018.08.003
ZHAO X, FENG J P, MENG S F. Considerations on Mycoplasma detection by nucleic acid detection method and the methodological validation. Chinese Pharmaceutical Affairs, 2018, 32(8):1020-1027. doi: 10.16153/j.1002-7777.2018.08.003. (in Chinese)
doi: 10.16153/j.1002-7777.2018.08.003
[7] ROTTEM S, BARILE M F. Beware of mycoplasmas. Trends in Biotechnology, 1993, 11(4):143-151. doi: 10.1016/0167-7799(93)90089-R.
doi: 10.1016/0167-7799(93)90089-R
[8] 中国兽药典委员会. 中华人民共和国兽药典-二部: 2015年版. 北京: 中国农业出版社, 2016.
Chinese Veterinary Pharmacopoeia Commission. Chinese Veterinary Pharmacopoeia of the People's Repubilic of China. Vol 2015. Beijing: Chinese Agriculture Press, 2016. (in Chinese)
[9] 国家药典委员会. 中华人民共和国药典-一部: 2020年版. 北京: 中国医药科技出版社, 2020.
Chinese Pharmacopoeia Commission. Chinese Pharmacopoeia of the People's Repubilic of China. Vol 2020. The Medicine Science and Technology Press of China, 2020. (in Chinese)
[10] DREXLER H G, UPHOFF C C. Mycoplasma contamination of cell cultures. Encyclopedia of Cell Technology, 2003. doi: 10.1002/0471250570.spi054.
doi: 10.1002/0471250570.spi054
[11] YOUNG L, SUNG J, STACEY G, MASTERS J R. Detection of Mycoplasma in cell cultures. Nature Protocols, 2010, 5(5):929-934. doi: 10.1038/nprot.2010.43.
doi: 10.1038/nprot.2010.43
[12] LIGASOVÁ A, VYDRŽALOVÁ M, BURIÁNOVÁ R, BRŮČKOVÁ L, VEČEŘOVÁ R, JANOŠŤÁKOVÁ A, KOBERNA K. A new sensitive method for the detection of mycoplasmas using fluorescence microscopy. Cells, 2019, 8(12):1510. doi: 10.3390/cells8121510.
doi: 10.3390/cells8121510
[13] BAI Y, GAN Y, HUA L Z, NATHUES H, YANG H, WEI Y N, WU M, SHAO G Q, FENG Z X. Application of a sIgA-ELISA method for differentiation of Mycoplasma hyopneumoniae infected from vaccinated pigs. Veterinary Microbiology, 2018, 223:86-92. doi: 10.1016/j.vetmic.2018.07.023.
doi: 10.1016/j.vetmic.2018.07.023
[14] QASEM J A, AL-MOUQATI S A, AL-ALI E M, BEN-HAJI A. Application of molecular and serological methods for rapid detection of Mycoplasma gallisepticum infection (Avian mycoplasmosis). Pakistan Journal of Biological Sciences: PJBS, 2015, 18(2):81-87. doi: 10.3923/pjbs.2015.81.87.
doi: 10.3923/pjbs.2015.81.87
[15] BEN ABDELMOUMEN MARDASSI B, BÉJAOUI A A, OUSSAEIF L, MLIK B, AMOUNA F. A recombinant antigen-based ELISA for the simultaneous differential serodiagnosis of Mycoplasma gallisepticum, Mycoplasma synoviae, and Mycoplasma meleagridis infections. Avian Diseases, 2008, 52(2):214-221. doi: 10.1637/8071-071207-Reg.1.
doi: 10.1637/8071-071207-Reg.1
[16] JOHANSSON K E, JOHANSSON I, GÖBEL U B. Evaluation of different hybridization procedures for the detection of mycoplasma contamination in cell cultures. Molecular and Cellular Probes, 1990, 4(1):33-42. doi: 10.1016/0890-8508(90)90037-Z.
doi: 10.1016/0890-8508(90)90037-Z
[17] KONG H, VOLOKHOV D V, GEORGE J, IKONOMI P, CHANDLER D, ANDERSON C, CHIZHIKOV V. Application of cell culture enrichment for improving the sensitivity of mycoplasma detection methods based on nucleic acid amplification technology (NAT). Applied Microbiology and Biotechnology, 2007, 77(1):223-232. doi: 10.1007/s00253-007-1135-1.
doi: 10.1007/s00253-007-1135-1
[18] UPHOFF C C, DREXLER H G. Detecting mycoplasma contamination in cell cultures by polymerase chain reaction. Methods in Molecular Biology (Clifton, N J), 2011, 731:93-103. doi: 10.1007/978-1-61779-080-5_8.
doi: 10.1007/978-1-61779-080-5_8
[19] 英聪, 王海光, 刘灿, 沈青春, 毕丁仁, 宁宜宝. 检测污染兽用疫苗14种支原体PCR方法的建立及应用. 中国预防兽医学报, 2014, 36(1):42-45. doi: 10.3969/j.issn.1008-0589.2014.01.11.
doi: 10.3969/j.issn.1008-0589.2014.01.11
YING C, WANG H G, LIU C, SHEN Q C, BI D R, NING Y B. The establishment of the PCR assay for detection of 14 kinds of Mycoplasma contamination in veterinary vaccines. Chinese Journal of Preventive Veterinary Medicine, 2014, 36(1):42-45. doi: 10.3969/j.issn.1008-0589.2014.01.11. (in Chinese)
doi: 10.3969/j.issn.1008-0589.2014.01.11
[20] INGEBRITSON A L, GIBBS C P, TONG C, SRINIVAS G B. A PCR detection method for testing mycoplasma contamination of veterinary vaccines and biological products. Letters in Applied Microbiology, 2015, 60(2):174-180. doi: 10.1111/lam.12355.
doi: 10.1111/lam.12355
[21] MBELO S, GAY V, BLANCHARD S, ABACHIN E, FALQUE S, LECHENET J, POULET H, DE SAINT-VIS B. Development of a highly sensitive PCR/DNA chip method to detect mycoplasmas in a veterinary modified live vaccine. Biologicals, 2018, 54:22-27. doi: 10.1016/j.biologicals.2018.05.002.
doi: 10.1016/j.biologicals.2018.05.002
[22] 高广仁, 万玉林, 崔治亮, 葛玉凤, 刘学荣. 兽用生物制品中支原体污染PCR检测方法的建立与应用. 黑龙江畜牧兽医, 2018(15):163-166. doi: 10.13881/j.cnki.hljxmsy.2017.12.0345.
doi: 10.13881/j.cnki.hljxmsy.2017.12.0345
GAO G R, WAN Y L, CUI Z L, GE Y F, LIU X R. Establishment and application of PCR for detection of mycoplasma contamination in veterinary biological products. Heilongjiang Animal Science and Veterinary Medicine, 2018(15):163-166. doi: 10.13881/j.cnki.hljxmsy.2017.12.0345. (in Chinese)
doi: 10.13881/j.cnki.hljxmsy.2017.12.0345
[23] ELDERING J A, FELTEN C, VEILLEUX C A, POTTS B J. Development of a PCR method for mycoplasma testing of Chinese hamster ovary cell cultures used in the manufacture of recombinant therapeutic proteins. Biologicals, 2004, 32(4):183-193. doi: 10.1016/j.biologicals.2004.08.005.
doi: 10.1016/j.biologicals.2004.08.005
[24] GRÓZNER D, SULYOK K M, KREIZINGER Z, RÓNAI Z, JÁNOSI S, TURCSÁNYI I, KÁROLYI H F, KOVÁCS Á B, KISS M J, VOLOKHOV D, GYURANECZ M. Detection of Mycoplasma anatis, M. anseris, M. cloacale and Mycoplasma sp. 1220 in waterfowl using species-specific PCR assays. PLoS ONE, 2019, 14(7):e0219071. doi: 10.1371/journal.pone.0219071.
doi: 10.1371/journal.pone.0219071
[25] WISSELINK H J, SMID B, PLATER J, RIDLEY A, ANDERSSON A M, ASPÁN A, POHJANVIRTA T, VÄHÄNIKKILÄ N, LARSEN H, HØGBERG J, COLIN A, TARDY F. A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis. BMC Veterinary Research, 2019, 15(1):86. doi: 10.1186/s12917-019-1819-7.
doi: 10.1186/s12917-019-1819-7
[26] TOTTEN A H, LEAL S M Jr, RATLIFF A E, XIAO L, CRABB D M, WAITES K B. Evaluation of the ELITe InGenius PCR platform for detection of Mycoplasma pneumoniae. Journal of Clinical Microbiology, 2019, 57(6):e00287-e00219. doi: 10.1128/JCM.00287-19.
doi: 10.1128/JCM.00287-19
[27] JEAN A, TARDY F, ALLATIF O, GROSJEAN I, BLANQUIER B, GERLIER D. Assessing mycoplasma contamination of cell cultures by qPCR using a set of universal primer pairs targeting a 1.5 kb fragment of 16S rRNA genes. PLoS ONE, 2017, 12(2):e0172358. doi: 10.1371/journal.pone.0172358.
doi: 10.1371/journal.pone.0172358
[28] BASCUÑANA C R, MATTSSON J G, BÖLSKE G, JOHANSSON K E. Characterization of the 16S rRNA genes from Mycoplasma sp. strain F38 and development of an identification system based on PCR. Journal of Bacteriology, 1994, 176(9):2577-2586. doi: 10.1128/jb.176.9.2577-2586.1994.
doi: 10.1128/jb.176.9.2577-2586.1994
[29] AMRAM E, MIKULA I, SCHNEE C, AYLING R D, NICHOLAS R A J, ROSALES R S, HARRUS S, LYSNYANSKY I. 16S rRNA gene mutations associated with decreased susceptibility to tetracycline in Mycoplasma bovis. Antimicrobial Agents and Chemotherapy, 2015, 59(2):796-802. doi: 10.1128/AAC.03876-14.
doi: 10.1128/AAC.03876-14
[30] BAKER G C, SMITH J J, COWAN D A. Review and re-analysis of domain-specific 16S primers. Journal of Microbiological Methods, 2003, 55(3):541-555. doi: 10.1016/j.mimet.2003.08.009.
doi: 10.1016/j.mimet.2003.08.009
[31] BAHRAM M, ANSLAN S, HILDEBRAND F, BORK P, TEDERSOO L. Newly designed 16S rRNA metabarcoding primers amplify diverse and novel archaeal taxa from the environment. Environmental Microbiology Reports, 2019, 11(4):487-494. doi: 10.1111/1758-2229.12684.
doi: 10.1111/1758-2229.12684
[32] 代玉立, 甘林, 滕振勇, 杨静民, 祁月月, 石妞妞, 陈福如, 杨秀娟. 玉米大斑病菌和小斑病菌交配型多重PCR检测方法的建立与应用. 中国农业科学, 2020, 53(3):527-538. doi: 10.3864/j.issn.0578-1752.2020.03.006.
doi: 10.3864/j.issn.0578-1752.2020.03.006
DAI Y L, GAN L, TENG Z Y, YANG J M, QI Y Y, SHI N N, CHEN F R, YANG X J. Establishment and application of a multiple PCR method to detect mating types of Exserohilum turcicum and Bipolaris maydis. Scientia Agricultura Sinica, 2020, 53(3):527-538. doi: 10.3864/j.issn.0578-1752.2020.03.006. (in Chinese)
doi: 10.3864/j.issn.0578-1752.2020.03.006
[33] 文静, 郭勇, 邱丽娟. 耐草甘膦转EPSPS/GAT大豆多重PCR检测体系的建立及应用. 中国农业科学, 2020, 53(20):4127-4136. doi: 10.3864/j.issn.0578-1752.2020.20.003.
doi: 10.3864/j.issn.0578-1752.2020.20.003
WEN J, GUO Y, QIU L J. Establishment and application of multiple PCR detection system for glyphosate-tolerant gene EPSPS/GAT in soybean. Scientia Agricultura Sinica, 2020, 53(20):4127-4136. doi: 10.3864/j.issn.0578-1752.2020.20.003. (in Chinese)
doi: 10.3864/j.issn.0578-1752.2020.20.003
[34] NIKFARJAM L, FARZANEH P. Prevention and detection of Mycoplasma contamination in cell culture. Cell Journal, 2012, 13(4):203-212.
[35] UPHOFF C C, DREXLER H G. Eradication of Mycoplasma contaminations from cell cultures. Current Protocols in Molecular Biology, 2014, 106:28.5.1-28.512. doi: 10.1002/0471142727.mb2805s106.
doi: 10.1002/0471142727.mb2805s106
[36] UPHOFF C C, DREXLER H G. Eradication of Mycoplasma contaminations. Basic Cell Culture Protocols, 2013, 946:15-26. doi: 10.1007/978-1-62703-128-8_2.
doi: 10.1007/978-1-62703-128-8_2
[37] UPHOFF C C, DREXLER H G. Detection of mycoplasma contaminations in cell cultures by PCR analysis. Human Cell, 1999, 12(4):229-236.
[38] KONG F, JAMES G, GORDON S, ZELYNSKI A, GILBERT G L. Species-specific PCR for identification of common contaminant mollicutes in cell culture. Applied and Environmental Microbiology, 2001, 67(7):3195-3200. doi: 10.1128/AEM.67.7.3195-3200.2001.
doi: 10.1128/AEM.67.7.3195-3200.2001
[39] 钱利武, 罗京京, 王浩, 刘小琼, 王慧. 药品GMP检查中质量控制与质量保证方面存在的主要问题及建议. 中国药事, 2020, 34(1):17-21. doi: 10.16153/j.1002-7777.2020.01.003.
doi: 10.16153/j.1002-7777.2020.01.003
QIAN L W, LUO J J, WANG H, LIU X Q, WANG H. Main problems of quality control and quality assurance in drug GMP inspection and the suggestions. Chinese Pharmaceutical Affairs, 2020, 34(1):17-21. doi: 10.16153/j.1002-7777.2020.01.003. (in Chinese)
doi: 10.16153/j.1002-7777.2020.01.003
[40] 谢寅, 刘晓萌, 吕建军, 孟建华, 王秀文. 瑞士GLP法令与中国GLP规范的主要差异. 中国药事, 2016, 30(4):352-354. doi: 10.16153/j.1002-7777.2016.04.008.
doi: 10.16153/j.1002-7777.2016.04.008
XIE Y, LIU X M, LÜ J J, MENG J H, WANG X W. Major differences between Swiss GLP articles and Chinese GLP. Chinese Pharmaceutical Affairs, 2016, 30(4):352-354. doi: 10.16153/j.1002-7777.2016.04.008. (in Chinese)
doi: 10.16153/j.1002-7777.2016.04.008
[1] LI FeiFei, LIAN XueFei, YIN Tao, CHANG YuanYuan, JIN Yan, MA XiaoChuan, CHEN YueWen, YE Li, LI YunSong, LU XiaoPeng. The Relationship Between Mastication and Development of Segment Membranes in Citrus Fruits [J]. Scientia Agricultura Sinica, 2023, 56(2): 333-344.
[2] ZHAI XiaoHu,LI LingXu,CHEN XiaoZhu,JIANG HuaiDe,HE WeiHua,YAO DaWei. Quantitative Detection Technology of Porcine-Derived Materials in Meat by Real-time PCR [J]. Scientia Agricultura Sinica, 2023, 56(1): 156-164.
[3] YANG XinRan,MA XinHao,DU JiaWei,ZAN LinSen. Expression Pattern of m6A Methylase-Related Genes in Bovine Skeletal Muscle Myogenesis [J]. Scientia Agricultura Sinica, 2023, 56(1): 165-178.
[4] WANG YiDan,YANG FaLong,CHEN DiShi,XIANG Hua,REN YuPeng. One-Step Multiple TaqMan Real-time RT-PCR for Simultaneous Detection of Swine Diarrhea Viruses [J]. Scientia Agricultura Sinica, 2023, 56(1): 179-192.
[5] LIU YuFang,CHEN YuLin,ZHOU ZuYang,CHU MingXing. miR-221-3p Regulates Ovarian Granulosa Cells Apoptosis by Targeting BCL2L11 in Small-Tail Han Sheep [J]. Scientia Agricultura Sinica, 2022, 55(9): 1868-1876.
[6] JIANG Hui,FENG Yu,QIN YuMing,ZHU LiangQuan,FAN XueZheng,DING JiaBo. Method Improvement and Its Application of Micro Complement Fixation Test for Brucellosis [J]. Scientia Agricultura Sinica, 2022, 55(8): 1676-1684.
[7] QIU YiLei,WU Fan,ZHANG Li,LI HongLiang. Effects of Sublethal Doses of Imidacloprid on the Expression of Neurometabolic Genes in Apis cerana cerana [J]. Scientia Agricultura Sinica, 2022, 55(8): 1685-1694.
[8] WANG SiTong,CHEN Yan,LUO YuJia,YANG YuanYuan,JIANG ZhiYang,JIANG XinYi,ZHONG Fan,CHEN Hao,XU HongXing,WU Yan,DUAN HongXia,TANG Bin. Effect of Three Novel Compounds on Trehalose and Chitin Metabolism and Development of Spodoptera frugiperda [J]. Scientia Agricultura Sinica, 2022, 55(8): 1568-1578.
[9] WU Yan,ZHANG Hao,LIANG ZhenHua,PAN AiLuan,SHEN Jie,PU YueJin,HUANG Tao,PI JinSong,DU JinPing. circ-13267 Regulates Egg Duck Granulosa Cells Apoptosis Through Let-7-19/ERBB4 Pathway [J]. Scientia Agricultura Sinica, 2022, 55(8): 1657-1666.
[10] WANG JiaMin,SHI JiaChen,MA FangFang,CAI Yong,QIAO ZiLin. Effects of Soy Isoflavones on the Proliferation and Apoptosis of Yak Ovarian Granulosa Cells [J]. Scientia Agricultura Sinica, 2022, 55(8): 1667-1675.
[11] PENG Xue,GAO YueXia,ZHANG LinXuan,GAO ZhiQiang,REN YaMei. Effects of High-Energy Electron Beam Irradiation on Potato Storage Quality and Bud Eye Cell Ultrastructure [J]. Scientia Agricultura Sinica, 2022, 55(7): 1423-1432.
[12] HE Lei,LU Kai,ZHAO ChunFang,YAO Shu,ZHOU LiHui,ZHAO Ling,CHEN Tao,ZHU Zhen,ZHAO QingYong,LIANG WenHua,WANG CaiLin,ZHU Li,ZHANG YaDong. Phenotypic Analysis and Gene Cloning of Rice Panicle Apical Abortion Mutant paa21 [J]. Scientia Agricultura Sinica, 2022, 55(24): 4781-4792.
[13] GUO ShaoLei,XU JianLan,WANG XiaoJun,SU ZiWen,ZHANG BinBin,MA RuiJuan,YU MingLiang. Genome-Wide Identification and Expression Analysis of XTH Gene Family in Peach Fruit During Storage [J]. Scientia Agricultura Sinica, 2022, 55(23): 4702-4716.
[14] XIE LiXue,ZHANG XiaoYan,ZHANG LiJie,ZHENG Shan,LI Tao. Complete Genome Sequence Characteristics and TC-RT-PCR Detection of East Asian Passiflora Virus Infecting Passiflora edulis [J]. Scientia Agricultura Sinica, 2022, 55(22): 4408-4418.
[15] CHEN Yu,ZHU HaoZhe,CHEN YiChun,LIU Zheng,DING Xi,GUO Yun,DING ShiJie,ZHOU GuangHong. Differentiation of Porcine Muscle Stem Cells in Three-Dimensional Hydrogels [J]. Scientia Agricultura Sinica, 2022, 55(22): 4500-4512.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd