绵羊肺腺瘤病毒的初步分离及PCR诊断的研究
摘要
绵羊肺腺瘤病是绵羊的一种传染性肺脏肿瘤病，由反转录病毒引起。本试验以某羊场病死羊肺脏作为病料，制成病理组织切片，通过病理组织学检查，结果表明：肺脏主要病变是肺泡细胞变为立方形或低柱状细胞，这些细胞沿肺泡壁排列，突入肺泡腔内形成乳头状腺瘤结构；在气管，支气管腔内有灰白色泡沫状液体，严重时使肺泡变为腺管状等。根据病理形态学特点，诊断为绵羊肺腺瘤病(SPA)。
将绵羊的胚胎肺细胞进行原代培养，接种绵羊肺腺瘤样品，进行病毒分离。接种后的胚肺细胞从第二代到第九代均出现细胞病变，当接毒细胞有3／4出现细胞病变时，收集培养的细胞，制作超薄切片进行电镜观察，发现了病毒样粒子。据报道，此病毒不易在体外培养，本试验对建立本病毒的体外培养体系进行了探索。
本试验还应用PCR技术，对培养的病毒进行检测；并对自然感染SPA的3例绵羊、随机采取的16例山羊和16例绵羊进行了诊断，结果表明：自然感染的3例绵羊确实患有SPA，为阳性；随机采取的16例绵羊中有2例为阳性，其余14例为阴性；16例山羊均为阴性，未感染SPA。通过此试验，对羊群是否患有SPA进行诊断，以建立一种SPA的特异性诊断方法。
Sheep pulmonary adenomatosis (synonyms, Jaagsiekte, ovine pulmonary carcinoma), a contagious lung cancer of sheep, was caused by an exogenous type D retrovirus, Jaagsiekte sheep retrovirus (JSRV). In this experiment, three dead sheep from SPA were been as materials to make histopathological and ultrastructural observation.
The results showed the primary lesion was the resultant transformation of type II epithelial alveolar cells. Tumors were composed mostly of single layers of cuboidal epithelial cells arranged in an acinar pattern, but all rumors had areas where the tumor cells piled up, forming papillary projections into the alveolar spaces. A striking and unique feature of SPA was the accumulation of fluid within the respiratory tract. According to the histopathological observation, diagnosed the case as sheep pulmonary adenomatosis.
In this experiment, the lung cells of embryo sheep were cultured and the cells were infected with JSRV. The infected cells were passaged so that the virus could be isolated. CPE were observed from passage 2 to passage 9 postinfection. The cultured virus supernatants were harvested when CPE were observed in 3/4 infected cells. The virions were found by electron-microscope observation. Studies on JSRV had been hampered by the lack of an infectious molecular JSRV clone and the lack of an in vitro infection system. An in vitro infection system in this experiment was studied.
In this experiment, the cultured virus was detected by PCR amplification. Moreover, 3 natural sheep cases, 16 sheep cases and 16 goat cases at random were diagnosed. The results showed 3 natural sheep cases were SPA, 2 of 16 sheep were positive and others were negative, 16 goats were negative. The herds were diagnosed whether they were sheep pulmonary adenomatosis by PCR in order to build up a specific method of diagnosing SPA in this experiment.
将绵羊的胚胎肺细胞进行原代培养，接种绵羊肺腺瘤样品，进行病毒分离。接种后的胚肺细胞从第二代到第九代均出现细胞病变，当接毒细胞有3／4出现细胞病变时，收集培养的细胞，制作超薄切片进行电镜观察，发现了病毒样粒子。据报道，此病毒不易在体外培养，本试验对建立本病毒的体外培养体系进行了探索。
本试验还应用PCR技术，对培养的病毒进行检测；并对自然感染SPA的3例绵羊、随机采取的16例山羊和16例绵羊进行了诊断，结果表明：自然感染的3例绵羊确实患有SPA，为阳性；随机采取的16例绵羊中有2例为阳性，其余14例为阴性；16例山羊均为阴性，未感染SPA。通过此试验，对羊群是否患有SPA进行诊断，以建立一种SPA的特异性诊断方法。
Sheep pulmonary adenomatosis (synonyms, Jaagsiekte, ovine pulmonary carcinoma), a contagious lung cancer of sheep, was caused by an exogenous type D retrovirus, Jaagsiekte sheep retrovirus (JSRV). In this experiment, three dead sheep from SPA were been as materials to make histopathological and ultrastructural observation.
The results showed the primary lesion was the resultant transformation of type II epithelial alveolar cells. Tumors were composed mostly of single layers of cuboidal epithelial cells arranged in an acinar pattern, but all rumors had areas where the tumor cells piled up, forming papillary projections into the alveolar spaces. A striking and unique feature of SPA was the accumulation of fluid within the respiratory tract. According to the histopathological observation, diagnosed the case as sheep pulmonary adenomatosis.
In this experiment, the lung cells of embryo sheep were cultured and the cells were infected with JSRV. The infected cells were passaged so that the virus could be isolated. CPE were observed from passage 2 to passage 9 postinfection. The cultured virus supernatants were harvested when CPE were observed in 3/4 infected cells. The virions were found by electron-microscope observation. Studies on JSRV had been hampered by the lack of an infectious molecular JSRV clone and the lack of an in vitro infection system. An in vitro infection system in this experiment was studied.
In this experiment, the cultured virus was detected by PCR amplification. Moreover, 3 natural sheep cases, 16 sheep cases and 16 goat cases at random were diagnosed. The results showed 3 natural sheep cases were SPA, 2 of 16 sheep were positive and others were negative, 16 goats were negative. The herds were diagnosed whether they were sheep pulmonary adenomatosis by PCR in order to build up a specific method of diagnosing SPA in this experiment.
引文
1 S. Kosati, M. pittall, A. Alberti, et al. An accessory open reading frame (orf—x) of Jaagsiekte sheep retrovirus is conserved between different virus isolates. Virus Research, 2000, 66:109—116
2 A Alexander, M Alley and J Conlon. New vaccines for the control of ovine pneumonia. 137—140
3 殷震，刘景华主编．动物病毒学(第二版)．北京：科学出版社，1997，222
4 A. J. Herring, J. M,Sharp, F. M. M. Scott, et al. Further evidence for a retrovirus as the aetiological agent of sheep pulmonary adenomatosis (Jaagsiekte). Veterinary Microbiology, 1983,8:237—249
5 K.Perk, R.Michalides, S. Spiegelman, et al. Biochemical and morphologic evidence for the presence of an RNA tumor virus in pulmonary carcinoma of sheep(Jaagsiekte). Journal of the National Cancer Institute, July 1974,53(1): 131—135
6 J. Bai, R. Y. Zhu, K. Stedman, et al. Unique long terminal repeat U_3 sequences distinguish exogenous Jaagsiekte sheep retroviruses associated with ovine pulmonary carcinoma from endogenous loci in the sheep genome. Journal of Virology, May 1996,3159—3168
7 M. Palmarini, P. Pewar, M. De las Heras, et al. Epithelial tumout cells in the lungs of sheep with pulmonary adenomatosis are major sites of replication for Jaagsiekte retrovirus. Journal of General Virology, 1995,76:2731—2737
8 Steven J. Hecht, Kim E. Stedman, Jonathan O. Carlson, et al. Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals. Medical Sciences, April 1996,93:3297—3302
9 L. Gonzalen, R. A. Juste, L. A. Cuervo, et al. Pathological and epidemiological aspects of the coexistence of maedi—visna and sheep pulmonary adenomatosis. Research in Veterinary Science, 1993, 54:140—146
10 M. Palmarini, C. Cousens, R. G. Dalziel, et al. The exogenous form of Jaagsiekte retrovirus is specifically associated with a contagious lung cancer of sheep. Journary of Virology, Mar 1996,1618—1623
11 R. H. Rosadio, J. M. Sharp, M. D. Lairmore, et al. Lesions and retroviruses associated with naturally occurring ovine pulmonary carcinoma (sheep pulmonary adenomatosis) .Vet. Pathol, 1988,25:58—66
12 Steven J. Hecht, Jonathan O. Carlson, and James C. Demartini. Analysis of a type D retroviral capsid gene expressed in ovine pulmonary carcinoma and present in both affected and unaffected sheep genomes. Virology 202, 1994, 480—484
13 邓普辉．简子健，刘君明等．绵羊肺腺瘤病的病原学研究．新疆畜牧，1993，1：26—29
14 Massimo Palmarini, J. Michael Sharp, Marcelode las Heras, et al. Jaagsiekte sheep retrovirus is necessary and sufficient to induce a contagious lung cancer in sheep. Journal of Virology, Aug
1999,73(8):6964—4972
15 Massimo Palmarini, J. Michael Sharp, Christine Lee, et al. In vitro infection of ovine cell lines by Jaagsiekte sheep retrovirus. Journal of Virology, Dec 1999,73(12): 10070—10078
16 邓普辉，Cutlip,R.C.中国和美国绵羊的肺腺瘤病．八一农学院学报，1986，9(3)：38—41
17 G. Di Guardc, R. Condoleo and G. L. Autorino. Multicentric lymphosarcoma associated with pulmonary adenomatosis(Jaagsiekte), pulmonary lymphoid hyperplasia, and lymphoid interstitial pneumonia in a Ewe. Vet. Pathol, 1992,29:262—264
18 F. J. Meyers, B. R. Madewell, P. H. Gumerlock, et al. Ras p21 expression in ovine pulmonary carcinoma. Veterinary Immunology and Immunopathology. 1989,23:279—291
19 Alzubaidy A.羊肺腺瘤病的研究．Indian Vet. 1979,56(5):360—362
20 J. M. Sharp and K. W. Angus. Sheep pulmonary adenomatosis:clinical, pathological and epidemiological aspects. Moredun Research Institute, Edinburgh Scotland. 58—174
21 张福萍，邓普辉，孟庆文等．自然感染的新疆美利奴羊中绵羊慢病毒的分离鉴定．中国畜禽传染病，1998，20(2)：68—70
22 邓普辉．绵羊肺腺瘤病一人细支气管—肺泡细胞癌的类似物．中国兽医科技，1988，2：58—59
23 邓普辉，简了键，白惠敏．新疆绵羊肺腺瘤病回顾性研究．中国兽医杂志，1996，22(1)：3—5
24 Experimental production of sheep pulmonary adenomatosis(Jaagsiekte). Nature Vol, Nov 1976,264
25 Denisf. York, Robert Vigne, Daniel W. Verwoerd, et al. Isolation. identification and partial cDNA cloning of genomic RNA of Jaagsiekte retrovirus, the etiological agent of sheep pulmonary, adenomatosis. Journal of Virology., Sept 1991,65(9):5061—5067
26 James C. Demartini, Jeanette V. Bishop, Thomas E. Allen, et al. Jaagsiekte sheep retrovirus proviral clone JSRV_(JS7), derived from the JS7 lung Tumour cell line, induces ovine pulmonary carcinoma and is integrated into the surfatant protein A gene. Journal of Virology, May 2001,4239—4246
27 Steven G. Irving, Kalman Perk, I Srael Hod, et al. Isolation and characterization of a novel retrovirus from sheep affected by pulmonary carcinoma. Virology 134,1984,244—248
28 Rosadio, R. H．与自我性绵羊肺癌(绵羊肺腺瘤病)伴发的病变与逆转录病毒．Veterinary Pathology,1988,25(1):58—66
29 L. Gonzalez, M. Garcia—Goti, C. Cousens, et al. Jaagsiekte sheep retrovirus can be detected in the peripheral blood during the pre—clinical period of sheep pulmonary adenomatosis. Journal of General Virology,2001,82:1355—1358
30 Osamu Kajikawa, John E. Dahlberg, Raul H. Rosadio, et al. Detection and quantitation of a type D retrovirus gag protein in ovine pulmonary carcinoma(sheep pulmonary adenomatosis)by means of a competition radioimmunoassay. Veterinary Microbiology, 1990,25:17—28
31 B. N. J. Parker, A. E. Wrathall, R. W. Saunders, et al. Prevention of transmission of sheep pulmonary
adenomatosis by embryo transfer. The Veterinary Record, June 1998,687—689
32 M. Dawson, S. H. Done, C.Venables, et al. Maedi—visna and sheep pulmonary adenomatosis: A study of concurrent infection. Bt. Vet. J. 1990,146,153
33 Denis F. York, Robert Vigne, Daniel W. Verwoerd, et al. Nucleotide sequence of the jaagsiekte retrovirus, and exogenous and endogenous type D and B retrovirus of sheep and goats. Journal of Virology, Aug 1992,4930—4939
34 Jirong Bai, Jeaneffe V. Bishop, Jonathan O. Carlson, et al. Sequence comparison of JSRV with endogenous proviruses:envelope genotypes and a novel ORF with similarity to a G-protein-coupled receptor. Virology 258,1999,333—343
35 Richard B. Whitney, Julia G. Levy, and Anajane G. Smith. Influence of tumour size and surgical resection on cell-mediated immunity in Mice. Journal of the National Cancer Institute, July 1974,53(1):111
36 李德宽．原发性黄牛肺腺癌病理形态观察，中国兽医杂志，1989，15(9)：40
37 赵松敏．绵羊肺腺瘤病综述．满洲里动植物检疫局，1—6
38 L. Hod, A. Zimber, U. Klopfer, et al. Pulmonary carcinoma(Jaagsiekte)of sheep:pathologic findings and comparison in multiple-case and case-free herds. Journal of the National Cancer Institute, July 1974,53(1):103—110
39 M. R. Alley. Pneumonio in sheep. Veterinary Annual 31,1991,S1—S8
40 邓普辉，张中庸，韩日衷等．新疆绵羊肺腺瘤病调查．中国兽医杂志，1981，7(11)：4—5
41 王选，洛桑江措，王晓丽．山羊肺腺瘤病的诊断．中国兽医杂志，1994，20(10)：18—19
42 Mullis.R.B. Scientific American, 1990,36—43
43 Mulllis. K. B. Usp, 1987,4:683
44 Saiki. R. K. et al. Science, 1985,230:1350—1354
45 Saiki. R. K. et al. Science, 1988,236:487—491
46 艾伯茨，布雷，约翰逊等著．基础细胞生物学——细胞分子生物学入门．赵寿元，金承志，丁小燕等译．上海：科学技术出版社，2002，304—307
47 阎隆飞，张玉麟主编．分子生物学(第二版)．中国农业大学出版社，2001，483
48 苏卫．聚合酶链反应及其在疾病诊断中的应用．中国兽医杂志，1991，17：7
49 冯作化．医学分子生物学．北京：人民卫生出版社，2001，184
50 Andrews AT. Electrophonesis, 1981,286
51 陈章良．PCR技术．北京大学，1991，10
52 Righy PWJ et al. Molecular Biological, 1977,113:237
53 K．B．穆里斯哥．聚合酶链式反应．陈受宜译．北京：科学出版社，1998，258—275
54 朱平．PCR基因扩增实验操作手册．中国科学技术出版社，1992
55 李文刚，甘孟候．PCR技术在兽医学中的应用．中国兽医杂志，1994，20(1)：53—54
56 翟中和，王喜忠，丁明孝．细胞生物学．高等教育出版社，2000，66
57 王伯，李玉松，黄高等主编．病理学技术．北京：人民卫生出版社，2002，796—807
58 叶俊华．警犬肺腺癌临床和病理观察．中国兽医杂志，1990，16(2)：27
59 林曦主编．家畜病理学(第三版)．中国农业出版社，283—284
60 甘肃农业大学主编．兽医微生物学实验指导．农业出版社，1980，30—41
61 G. C. Pritchard, S. H. Done. Concurrent maedi-visna virus infection and pulmonary adenomatosis in a commercial breeding flock in East Anglia. Veterinary Record, 1990,127:197—200
62 J．萨姆布鲁克，E．F．弗里奇等著．分子克隆实验指南(第二版)．金冬雁等译．科学出版社，1995
2 A Alexander, M Alley and J Conlon. New vaccines for the control of ovine pneumonia. 137—140
3 殷震，刘景华主编．动物病毒学(第二版)．北京：科学出版社，1997，222
4 A. J. Herring, J. M,Sharp, F. M. M. Scott, et al. Further evidence for a retrovirus as the aetiological agent of sheep pulmonary adenomatosis (Jaagsiekte). Veterinary Microbiology, 1983,8:237—249
5 K.Perk, R.Michalides, S. Spiegelman, et al. Biochemical and morphologic evidence for the presence of an RNA tumor virus in pulmonary carcinoma of sheep(Jaagsiekte). Journal of the National Cancer Institute, July 1974,53(1): 131—135
6 J. Bai, R. Y. Zhu, K. Stedman, et al. Unique long terminal repeat U_3 sequences distinguish exogenous Jaagsiekte sheep retroviruses associated with ovine pulmonary carcinoma from endogenous loci in the sheep genome. Journal of Virology, May 1996,3159—3168
7 M. Palmarini, P. Pewar, M. De las Heras, et al. Epithelial tumout cells in the lungs of sheep with pulmonary adenomatosis are major sites of replication for Jaagsiekte retrovirus. Journal of General Virology, 1995,76:2731—2737
8 Steven J. Hecht, Kim E. Stedman, Jonathan O. Carlson, et al. Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals. Medical Sciences, April 1996,93:3297—3302
9 L. Gonzalen, R. A. Juste, L. A. Cuervo, et al. Pathological and epidemiological aspects of the coexistence of maedi—visna and sheep pulmonary adenomatosis. Research in Veterinary Science, 1993, 54:140—146
10 M. Palmarini, C. Cousens, R. G. Dalziel, et al. The exogenous form of Jaagsiekte retrovirus is specifically associated with a contagious lung cancer of sheep. Journary of Virology, Mar 1996,1618—1623
11 R. H. Rosadio, J. M. Sharp, M. D. Lairmore, et al. Lesions and retroviruses associated with naturally occurring ovine pulmonary carcinoma (sheep pulmonary adenomatosis) .Vet. Pathol, 1988,25:58—66
12 Steven J. Hecht, Jonathan O. Carlson, and James C. Demartini. Analysis of a type D retroviral capsid gene expressed in ovine pulmonary carcinoma and present in both affected and unaffected sheep genomes. Virology 202, 1994, 480—484
13 邓普辉．简子健，刘君明等．绵羊肺腺瘤病的病原学研究．新疆畜牧，1993，1：26—29
14 Massimo Palmarini, J. Michael Sharp, Marcelode las Heras, et al. Jaagsiekte sheep retrovirus is necessary and sufficient to induce a contagious lung cancer in sheep. Journal of Virology, Aug
1999,73(8):6964—4972
15 Massimo Palmarini, J. Michael Sharp, Christine Lee, et al. In vitro infection of ovine cell lines by Jaagsiekte sheep retrovirus. Journal of Virology, Dec 1999,73(12): 10070—10078
16 邓普辉，Cutlip,R.C.中国和美国绵羊的肺腺瘤病．八一农学院学报，1986，9(3)：38—41
17 G. Di Guardc, R. Condoleo and G. L. Autorino. Multicentric lymphosarcoma associated with pulmonary adenomatosis(Jaagsiekte), pulmonary lymphoid hyperplasia, and lymphoid interstitial pneumonia in a Ewe. Vet. Pathol, 1992,29:262—264
18 F. J. Meyers, B. R. Madewell, P. H. Gumerlock, et al. Ras p21 expression in ovine pulmonary carcinoma. Veterinary Immunology and Immunopathology. 1989,23:279—291
19 Alzubaidy A.羊肺腺瘤病的研究．Indian Vet. 1979,56(5):360—362
20 J. M. Sharp and K. W. Angus. Sheep pulmonary adenomatosis:clinical, pathological and epidemiological aspects. Moredun Research Institute, Edinburgh Scotland. 58—174
21 张福萍，邓普辉，孟庆文等．自然感染的新疆美利奴羊中绵羊慢病毒的分离鉴定．中国畜禽传染病，1998，20(2)：68—70
22 邓普辉．绵羊肺腺瘤病一人细支气管—肺泡细胞癌的类似物．中国兽医科技，1988，2：58—59
23 邓普辉，简了键，白惠敏．新疆绵羊肺腺瘤病回顾性研究．中国兽医杂志，1996，22(1)：3—5
24 Experimental production of sheep pulmonary adenomatosis(Jaagsiekte). Nature Vol, Nov 1976,264
25 Denisf. York, Robert Vigne, Daniel W. Verwoerd, et al. Isolation. identification and partial cDNA cloning of genomic RNA of Jaagsiekte retrovirus, the etiological agent of sheep pulmonary, adenomatosis. Journal of Virology., Sept 1991,65(9):5061—5067
26 James C. Demartini, Jeanette V. Bishop, Thomas E. Allen, et al. Jaagsiekte sheep retrovirus proviral clone JSRV_(JS7), derived from the JS7 lung Tumour cell line, induces ovine pulmonary carcinoma and is integrated into the surfatant protein A gene. Journal of Virology, May 2001,4239—4246
27 Steven G. Irving, Kalman Perk, I Srael Hod, et al. Isolation and characterization of a novel retrovirus from sheep affected by pulmonary carcinoma. Virology 134,1984,244—248
28 Rosadio, R. H．与自我性绵羊肺癌(绵羊肺腺瘤病)伴发的病变与逆转录病毒．Veterinary Pathology,1988,25(1):58—66
29 L. Gonzalez, M. Garcia—Goti, C. Cousens, et al. Jaagsiekte sheep retrovirus can be detected in the peripheral blood during the pre—clinical period of sheep pulmonary adenomatosis. Journal of General Virology,2001,82:1355—1358
30 Osamu Kajikawa, John E. Dahlberg, Raul H. Rosadio, et al. Detection and quantitation of a type D retrovirus gag protein in ovine pulmonary carcinoma(sheep pulmonary adenomatosis)by means of a competition radioimmunoassay. Veterinary Microbiology, 1990,25:17—28
31 B. N. J. Parker, A. E. Wrathall, R. W. Saunders, et al. Prevention of transmission of sheep pulmonary
adenomatosis by embryo transfer. The Veterinary Record, June 1998,687—689
32 M. Dawson, S. H. Done, C.Venables, et al. Maedi—visna and sheep pulmonary adenomatosis: A study of concurrent infection. Bt. Vet. J. 1990,146,153
33 Denis F. York, Robert Vigne, Daniel W. Verwoerd, et al. Nucleotide sequence of the jaagsiekte retrovirus, and exogenous and endogenous type D and B retrovirus of sheep and goats. Journal of Virology, Aug 1992,4930—4939
34 Jirong Bai, Jeaneffe V. Bishop, Jonathan O. Carlson, et al. Sequence comparison of JSRV with endogenous proviruses:envelope genotypes and a novel ORF with similarity to a G-protein-coupled receptor. Virology 258,1999,333—343
35 Richard B. Whitney, Julia G. Levy, and Anajane G. Smith. Influence of tumour size and surgical resection on cell-mediated immunity in Mice. Journal of the National Cancer Institute, July 1974,53(1):111
36 李德宽．原发性黄牛肺腺癌病理形态观察，中国兽医杂志，1989，15(9)：40
37 赵松敏．绵羊肺腺瘤病综述．满洲里动植物检疫局，1—6
38 L. Hod, A. Zimber, U. Klopfer, et al. Pulmonary carcinoma(Jaagsiekte)of sheep:pathologic findings and comparison in multiple-case and case-free herds. Journal of the National Cancer Institute, July 1974,53(1):103—110
39 M. R. Alley. Pneumonio in sheep. Veterinary Annual 31,1991,S1—S8
40 邓普辉，张中庸，韩日衷等．新疆绵羊肺腺瘤病调查．中国兽医杂志，1981，7(11)：4—5
41 王选，洛桑江措，王晓丽．山羊肺腺瘤病的诊断．中国兽医杂志，1994，20(10)：18—19
42 Mullis.R.B. Scientific American, 1990,36—43
43 Mulllis. K. B. Usp, 1987,4:683
44 Saiki. R. K. et al. Science, 1985,230:1350—1354
45 Saiki. R. K. et al. Science, 1988,236:487—491
46 艾伯茨，布雷，约翰逊等著．基础细胞生物学——细胞分子生物学入门．赵寿元，金承志，丁小燕等译．上海：科学技术出版社，2002，304—307
47 阎隆飞，张玉麟主编．分子生物学(第二版)．中国农业大学出版社，2001，483
48 苏卫．聚合酶链反应及其在疾病诊断中的应用．中国兽医杂志，1991，17：7
49 冯作化．医学分子生物学．北京：人民卫生出版社，2001，184
50 Andrews AT. Electrophonesis, 1981,286
51 陈章良．PCR技术．北京大学，1991，10
52 Righy PWJ et al. Molecular Biological, 1977,113:237
53 K．B．穆里斯哥．聚合酶链式反应．陈受宜译．北京：科学出版社，1998，258—275
54 朱平．PCR基因扩增实验操作手册．中国科学技术出版社，1992
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