牛羊布鲁氏菌天然半抗原琼脂扩散试验的建立与应用

doi:10.6048/j.issn.1001-4330.2024.08.027

新疆农业科学 ›› 2024, Vol. 61 ›› Issue (8): 2063-2070.DOI: 10.6048/j.issn.1001-4330.2024.08.027

• 植物保护·设施农业·畜牧兽医·农业经济 • 上一篇下一篇

牛羊布鲁氏菌天然半抗原琼脂扩散试验的建立与应用

刘丽娅¹(), 剡文亮², 曹瑞³, 叶锋¹, 马晓菁¹, 谷文喜¹, 赵江山⁴, 张子荣⁵, 宋洁⁶, 李岩²(), 易新萍¹()

1.新疆动物疫病研究重点实验室/新疆畜牧科学院兽医研究所,乌鲁木齐 830011
2.新疆生产建设兵团畜牧兽医工作总站,乌鲁木齐 830021
3.青岛瑞尔唯特生物技术有限公司,山东青岛 266000
4.新疆维吾尔自治区疾病预防控制中心,乌鲁木齐 830000
5.新疆生产建设兵团第六师畜牧兽医工作站,新疆五家渠 831304
6.新疆天润乳业股份有限公司,乌鲁木齐 830000

收稿日期:2024-01-18 出版日期:2024-08-20 发布日期:2024-09-19
通信作者: 易新萍(1970-),女,湖北公安人,研究员,博士,研究方向为动物疫病防控,(E-mail)821489803@qq.com;
李岩(1966-),男,山东人,研究员,硕士生导师,研究方向为动物疫病预防控制,(E-mail)bt4641862@163.com
作者简介:刘丽娅(1984-),女,安徽阜阳人,副研究员,硕士,研究方向为动物疫病防控,(E-mail)286238083@qq.com
基金资助:
新疆维吾尔自治区重点研发专项(2022B03013-1);新疆生产建设兵团重点领域科技攻关计划项目(2020AB015)

Establishment and application of natural hapten agar diffusion test for brucella

LIU Liya¹(), YAN Wenliang², CAO Rui³, YE Feng¹, MA Xiaojing¹, GU Wenxi¹, ZHAO Jiangshan⁴, ZHANG Zirong⁵, SONG Jie⁶, LI Yan²(), YI Xinping¹()

1. Xinjiang Key Laboratory of Animal Infectious Diseases, Institute of Veterinary Medicine, Xinjang Academy of Animal Sciences, Urumqi 830011,China
2. The General Station of Animal Husbandry and Veterinary of Xinjiang Production and Construction Corps, Urumqi 830021, China
3. Qingdao Real Bio-Technology Co., Ltd, Qingdao Shandong 266000, China
4. Centre for Disease Control and Prevention of Xinjiang Uygur Autonomous Region, Urumqi 830000,China
5. Animal Husbandry and Veterinary Work Station of the Sixth Division, Xinjiang Production and Construction Corps, Wujiaqu Xinjiang 831304,China
6. Xinjiang Tianrun Dairy limited Co.,Ltd, Urumqi 830000, China

Received:2024-01-18 Published:2024-08-20 Online:2024-09-19
Correspondence author: YI Xinping(1970-), female, from Gongan, Hubei, PhD, researcher, research direction: animal disease prevention and control research, (E-mail)821489803@qq.com;
LI Yan(1966-), male, from Shandong, researcher,Master's supervisor, research direction: animal disease prevention and control, (E-mail)bt4641862@163.com
Supported by:
Key R&D Special Project of Xinjiang Uygur Autonomous Region(2022B03013-1);The Project of Tackling Hard-Nut Problems in Science and Technology of XPCC(2020AB015)

摘要/Abstract

摘要：

【目的】建立并优化布鲁氏菌天然半抗原琼脂扩散试验(Native hapten agar gel immuno-diffusion test,NH-AGID),检测临床样品。【方法】建立NH-AGID方法,评价方法特异性、敏感性及重复性;检测免疫地区2 287份、非免疫地区252份牛、羊血清。【结果】建立了NH-AGID方法,检测稳定,重复性好,具有较高的特异性;对自然感染阳性动物检测敏感性低于80%,对排菌动物的检测敏感性高于90%,检出布病阳性抗体的阈值高于RBT。免疫地区血清NH抗体平均检出率17.8%,LPS抗体平均检出率52.3%;非免疫地区血清NH抗体平均检出率40.9%,LPS抗体平均检出率54.8%。【结论】应用NH-AGID方法可鉴别区分免疫地区牛、羊布病感染动物和免疫动物。检出NH抗体动物处于布鲁氏菌活动期或排菌期为布病感染动物,应及时剔除出群。

关键词: 布鲁氏菌病; 半抗原-琼脂扩散试验; 诊断

Abstract:

【Objective】 To establish and optimize the natural hapten agar gel immuno-diffusion test (NH-AGID) for Brucella abortus in clinical samples.【Methods】 The NH-AGID method was established to evaluate the specificity, sensitivity and reproducibility of the method; 2,287 bovine and sheep sera from immunized areas and 252 from non-immunized areas were tested. 【Results】 The NH-AGID method with stable and reproducible assay and high specificity was successfully established; The sensitivity of the assay for naturally infected positive animals was lower than 80%; The sensitivity of the assay for releasing animals was higher than 90%; The threshold for detecting bruclosis-positive antibodies was higher than RBT. The average detection rate of serum NH antibodies in immunized areas was 17.8%, and the average detection rate of LPS antibodies was 52.3%; the average detection rate of serum NH antibodies in non-immunized areas was 40.9%, and the average detection rate of LPS antibodies was 54.8%. 【Conclusion】 The NH-AGID method can be applied to identify and distinguish bovine and sheep brucellosis-infected animals from immunized animals in immunized areas. Animals detected with NH antibodies in the active or excretory phase of Brucella abortus are brucellosis-infected animals and should be removed from the herd in time.

Key words: brucellosis; native hapten-agar gel immuno diffusion; diagnosis

中图分类号:

S855

刘丽娅, 剡文亮, 曹瑞, 叶锋, 马晓菁, 谷文喜, 赵江山, 张子荣, 宋洁, 李岩, 易新萍. 牛羊布鲁氏菌天然半抗原琼脂扩散试验的建立与应用[J]. 新疆农业科学, 2024, 61(8): 2063-2070.

LIU Liya, YAN Wenliang, CAO Rui, YE Feng, MA Xiaojing, GU Wenxi, ZHAO Jiangshan, ZHANG Zirong, SONG Jie, LI Yan, YI Xinping. Establishment and application of natural hapten agar diffusion test for brucella[J]. Xinjiang Agricultural Sciences, 2024, 61(8): 2063-2070.

图/表 10

图1 图1 NH-AGID方法

Fig.1 Result display diagram of NH-AGID method

表1 布鲁氏菌NH-AGID方法最佳抗原浓度

Tab.1 Best antigen concentration test of Brucella NH-AGID

血清 Serum	不同抗原浓度 Different antigen concentrations
	0.5 (mg/mL)		1.0 (mg/mL)		1.5 (mg/mL)		2.0 (mg/mL)		2.5 (mg/mL)		3.0 (mg/mL)
	NH	LPS	NH	LPS	NH	LPS	NH	LPS	NH	LPS	NH	LPS
牛强阳 Cattle strong positive serum	+	+	+	+	+	+	+	+	+	+	+	+
羊强阳 Sheep strong positive serum	+	+	+	+	+	+	+	+	-	+	+	+
牛中阳 Positive serum in cattle	+	+	+	+	+	+	+	+	+	+	-	-
羊中阳 Positive serum in sheep	+	+	+	+	+	+	-	+	+	+	-	-
牛弱阳 Cattle weakly positive serum	+	+	+	+	+	+	+	+	-	-	-	-
羊弱阳 Sheep weakly positive serum	-	+	+	+	+	+	-	+	-	+	+	+

表2 布鲁氏菌NH-AGID方法最佳琼脂平板浓度

Tab.2 The best agar plate concentration test of Brucella NH-AGID

血清 Serum	不同琼脂糖浓度 Different agarose concentrations
	0.6%		1.0%		1.2%		1.5%		1.7%		2.0%
	NH	LPS	NH	LPS	NH	LPS	NH	LPS	NH	LPS	NH	LPS
牛强阳 Cattle strong positive serum	+	+	+	+	+	+	+	+	+	+	+	+
羊强阳 Sheep strong positive serum	+	+	+	+	+	+	+	+	+	+	+	+
牛中阳 Positive serum in cattle	+	+	+	+	+	+	+	+	+	+	+	+
羊中阳 Positive serum in sheep	+	+	+	+	+	+	+	+	+	-	+	-
牛弱阳 Cattle weakly positive serum	-	+	+	+	+	+	+	-	-	-	-	-
羊弱阳 Sheep weakly positive serum	-	-	-	+	+	+	-	-	-	-	-	-

表3 布鲁氏菌NH-AGID方法最佳最佳孵育温度和孵育时间

Tab.3 Experiment on the best incubation temperature and time of Brucella NH-AGID

血清 Serum	孵育时间 Incubation time (h)	不同孵育温度 Different incubation temperatures
		18℃		22℃		25℃		30℃		37℃
		NH	LPS	NH	LPS	NH	LPS	NH	LPS	NH	LPS
牛强阳 Cattle strong positive serum	24	-	+	+	+	+	+	+	+	+	+
羊强阳 Sheep strong positive serum	24	+	+	+	+	+	+	+	+	+	+
牛中阳 Positive serum in cattle	24	-	+	+	+	+	+	+	+	+	+
羊中阳 Positive serum in sheep	24	+	+	+	+	+	+	+	+	+	+
牛弱阳 Cattle weakly positive serum	24	-	-	+	+	+	+	+	+	+	+
羊弱阳 Sheep weakly positive serum	24	-	-	-	+	+	+	+	+	+	+

表4 布鲁氏菌NH-AGID与RBT检测敏感性差异

Tab.4 Sensitivity difference test of Brucella NH-AGID and RBT detection

强阳性血清稀释倍数 Strongly positive serum dilution multiplier	RBT结果 RBT Result	检测结果 Test results
强阳性血清稀释倍数 Strongly positive serum dilution multiplier	RBT结果 RBT Result	NH	LPS
1:2	++++	+	+
1:4	++++	+	+
1:8	+++	+	+
1:16	+++	-	+
1:32	++	-	+
1:64	++	-	-
1:128	-	-	-
1:256	-	-	-

表5 自然感染动物布鲁氏菌NH-AGID方法检测结果

Tab.5 Detection results of Brucella NH-AGID natural infection animals

动物种类 Animal species	检测数 Number of tests (copies)	RBT 检测 RBT Detection	SAT 检测 SAT Detection	NH-AGID
				NH & LPS		LPS
				检出数 Number of detections (copies)	检出率 Detection rate (%)	检出数 Number of detections (copies)	检出率 Detection rate (%)
牛 Cattle	50	+	+	34	68.0	44	88.0
羊 Sheep	50	+	+	38	76.0	47	94.0
合计 Total	100	+	+	72	72.0	91	91.0

表6 PCR检测阳性动物布鲁氏菌NH-AGID方法检测结果

Tab.6 Detection results of NH-AGID for Brucella in animals with positive PCR detection

动物种类 Animal species	检测数 Number of tests (copies)	RBT 检测 RBT Detection	PCR 检测 PCR Detection	NH-AGID
				NH & LPS		LPS
				检出数 Number of detections (copies)	检出率 Detection rate (%)	检出数 Number of detections (copies)	检出率 Detection rate (%)
牛 Cattle	24	+	+	22	91.7	24	100.0
羊 Sheep	24	+	+	23	95.8	24	100.0
合计 Total	48	+	+	45	93.8	48	100.0

表7 布鲁氏菌NH-AGID方法特异性

Tab.7 Specificity test of Brucella NH-AGID

血清 Serum	检测结果 Test results
血清 Serum	检测数 Number of tests (copies)	NH NH (copies)	LPS LPS(copies)
口蹄疫O型抗体阳性血清 Foot-and-mouth disease O antibody-positive serum	3	0	0
口蹄疫A型抗体阳性血清 Foot-and-mouth disease A antibody-positive serum	4	0	0
牛支原体抗体阳性血清 Bovine mycoplasma antibody-positive serum	5	0	0
牛分枝杆菌抗体阳性血清 Bovine Mycobacterium antibodies positive serum	5	0	0
大肠杆菌抗体阳性血清 E. coli antibody-positive serum	10	0	0
牛布病阴性血清 Bovine brucellosis negative serum	20	0	0
羊布病阴性血清 Sheep brucellosis negative serum	20	0	0

表8 布鲁氏菌NH-AGID方法免疫地区血清样品检测

Tab.8 Detection results of Brucella in serum samples from immune areas by NH-AGID

采集地区 Collection area	血清类型 Serum Type	检测数 Number of tests (copies)	RBT 检测 RBT Detection	NH-AGID
				NH & LPS		LPS
				阳性数 Number of positive (copies)	阳性率 Positive rate (%)	阳性数 Number of positive (copies)	阳性率 Positive rate (%)
免疫地区 Immunized areas	A19- $△$ VirB12株免疫牛血清	342	+	19	5.6	269	78.7
	A19株免疫牛血清	1688	+	313	18.5	784	46.4
	M5株免疫羊血清	257	+	74	28.8	142	55.3
合计 Total		2287	+	406	17.8	1195	52.3

表8 布鲁氏菌NH-AGID方法免疫地区血清样品检测

Tab.8 Detection results of Brucella in serum samples from immune areas by NH-AGID

采集地区 Collection area	血清类型 Serum Type	检测数 Number of tests (copies)	RBT 检测 RBT Detection	NH-AGID
				NH & LPS		LPS
				阳性数 Number of positive (copies)	阳性率 Positive rate (%)	阳性数 Number of positive (copies)	阳性率 Positive rate (%)
免疫地区 Immunized areas	A19- $△$ VirB12株免疫牛血清	342	+	19	5.6	269	78.7
	A19株免疫牛血清	1688	+	313	18.5	784	46.4
	M5株免疫羊血清	257	+	74	28.8	142	55.3
合计 Total		2287	+	406	17.8	1195	52.3

表9 布鲁氏菌NH-AGID方法非免疫地区血清样品检测

Tab.9 Detection results of Brucella in serum samples from non immune areas by NH-AGID

采集地区 Collection area	血清类型 Serum Type	检测数 Number of tests (copies)	SAT 检测 SAT Detection	NH-AGID
				NH & LPS		LPS
				阳性数 Number of positive (copies)	阳性率 Positive rate (%)	阳性数 Number of positive (copies)	阳性率 Positive rate (%)
非免疫地区 Non-immunized areas	牛	55	+	19	34.5	33	60.0
非免疫地区 Non-immunized areas	羊	197	+	84	42.6	105	53.3
合计 Total		252	+	103	40.9	138	54.8

参考文献 21

[1]	赵婷婷, 赵世刚, 苏霄, 等. 神经型布鲁氏菌病的发病机制、诊断方法及预防[J]. 中国人兽共患病学报, 2020, 36(6): 503-508.
	ZHAO Tingting, ZHAO Shigang, SU Xiao, et al. Pathogenesis, diagnosis and prevention of neuropathic brucellosis[J]. Chinese Journal of Zoonoses, 2020, 36(6): 503-508.
[2]	Lai S J, Zhou H, Xiong W Y, et al. Changing epidemiology of human brucellosis, China, 1955-2014[J]. Emerging Infectious Diseases, 2017, 23(2): 184-194. DOI PMID
[3]	Nurpeisova A K, Kolomeietz A N. The perspectives of studying of polymorphism of genes of gamma-interferon under chronic brucellosis[J]. Klinicheskaia Laboratornaia Diagnostika, 2016, 61(2): 110-113.
[4]	汪洁英, 宁博, 景伟, 等. 布鲁氏菌病及其在我国的防控现状与建议[J]. 中国兽医科学, 2022, 52(12): 1578-1585.
	WANG Jieying, NING Bo, JING Wei, et al. Research progress and suggestions regarding on the prevention and control of brucellosis in China: a review[J]. Chinese Veterinary Science, 2022, 52(12): 1578-1585.
[5]	刘志国, 王妙, 崔步云, 等. 布鲁氏菌胞内存活及疫苗研究进展[J]. 中国人兽共患病学报, 2019, 35(5): 430-439.
	LIU Zhiguo, WANG Miao, CUI Buyun, et al. Brucella intracellular survival and vaccine research progress[J]. Chinese Journal of Zoonoses, 2019, 35(5): 430-439.
[6]	景志刚, 严家瑞, 范伟兴. 布鲁氏菌病疫苗研究进展[J]. 中国人兽共患病学报, 2016, 32(2): 188-199.
	JING Zhigang, YAN Jiarui, FAN Weixing. Research progress of brucellosis vaccines[J]. Chinese Journal of Zoonoses, 2016, 32(2): 188-199.
[7]	朱一帆, 陈颖钰, 胡长敏, 等. 我国牛重要传染病流行现状和防控建议[J/OL]. 华中农业大学学报, 2022: 1-8.
	ZHU Yifan, CHEN Yingyu, HU Changmin, et al. Epidemiological status and prevention and control suggestion of important bovine infectious diseases in China[J/OL]. Journal of Huazhong Agricultural University, 2022: 1-8.
[8]	Marín C M, Moreno E, Moriyón I, et al. Performance of competitive and indirect enzyme-linked immunosorbent assays, gel immunoprecipitation with native hapten polysaccharide, and standard serological tests in diagnosis of sheep brucellosis[J]. Clinical and Diagnostic Laboratory Immunology, 1999, 6(2): 269-272. DOI PMID
[9]	Jones L M, Berman D T, Moreno E, et al. Evaluation of a radial immunodiffusion test with polysaccharide B antigen for diagnosis of bovine brucellosis[J]. Journal of Clinical Microbiology, 1980, 12(6): 753-760. DOI PMID
[10]	景添, 王天星, 陈曦, 等. 羊布鲁氏菌病及其国内外防控净化措施[J]. 动物医学进展, 2022, 43(2): 116-120.
	JING Tian, WANG Tianxing, CHEN Xi, et al. Control and eradication of brucellosis in goats and sheep: experiences learned from China and other countries[J]. Progress in Veterinary Medicine, 2022, 43(2): 116-120.
[11]	张军, 黄宇翔, 王志强, 等. 影响琼脂扩散试验因素分析[J]. 畜禽业, 2019, 30(8): 14.
	ZHANG Jun, HUANG Yuxiang, WANG Zhiqiang, et al. Analysis of factors affecting agar diffusion test[J]. Livestock and Poultry Industry, 2019, 30(8): 14.
[12]	王金涛, 陈国福, 刘光前. 动物布鲁氏菌病免疫学诊断方法研究进展[J]. 现代化农业, 2022,(1): 2-4.
	WANG Jintao, CHEN Guofu, LIU Guangqian. Research progress on immunological diagnosis methods of animal brucellosis[J]. Modernizing Agriculture, 2022,(1): 2-4.
[13]	Bundle D R, Cherwonogrodzky J W, Perry M B. Characterization of Brucella polysaccharide B[J]. Infection and Immunity, 1988, 56(5): 1101-1106. DOI PMID
[14]	Aragón V, Díaz R, Moreno E, et al. Characterization of Brucella abortus and Brucella melitensis native haptens as outer membrane O-type polysaccharides independent from the smooth lipopolysaccharide[J]. Journal of Bacteriology, 1996, 178(4): 1070-1079. PMID
[15]	梅建军, 石慧英, 王兴龙. 布鲁氏菌表面抗原研究进展[J]. 动物医学进展, 2005, 26(10): 13-18.
	MEI Jianjun, SHI Huiying, WANG Xinglong. Progress on surface antigens of Brucella spp[J]. Progress In Veterinary Medicine, 2005, 26(10): 13-18.
[16]	Forsyth J R L. Joint FAO/WHO expert committee on brucellosis joint FAO/WHO expert committee on brucellosis. World Health Organization, Geneva, 1986. ISBN 92-4-120740-X, pp. 132. US$10.80[J]. Pathology, 1987, 19(4): 437.
[17]	杨珍, 尼博, 田莉莉, 等. 半抗原-琼脂扩散试验鉴别布鲁氏菌感染抗体和S2、Rev.1免疫抗体的研究与应用[J]. 中国人兽共患病学报, 2017, 33(2): 126-130.
	YANG Zhen, NI Bo, TIAN Lili, et al. Application of native hapten-gel diffusion test in differential diagnosis of Brucella-infected antibody and S2 or Rev.1-vaccinated antibody[J]. Chinese Journal of Zoonoses, 2017, 33(2): 126-130.
[18]	Forsyth J R L. Joint FAO/WHO expert committee on brucellosis[J]. Pathology, 1987, 19(4): 437.
[19]	孙晶晶, 吴锦艳, 曹小安, 等. 布鲁氏菌S2、M5及A19疫苗免疫羊抗体消长规律研究[J]. 中国预防兽医学报, 2020, 42(3): 287-292.
	SUN Jingjing, WU Jinyan, CAO Xiaoan, et al. Study on the growth and decline of antibody against Brucella S2, M5 and A19 vaccine in immunized sheep[J]. Chinese Journal of Preventive Veterinary Medicine, 2020, 42(3): 287-292.
[20]	易新萍, 谷文喜, 吴冬玲, 等. 流产布鲁氏菌疫苗A19-ΔVirB12突变株生物学特性研究[J]. 中国人兽共患病学报, 2013, 29(9): 836-840.
	YI Xinping, GU Wenxi, WU Dongling, et al. Biological characteristics of vaccine candidate A19-ΔVirB12 mutant strain of Brucella abortus[J]. Chinese Journal of Zoonoses, 2013, 29(9): 836-840.
[21]	易新萍, 叶锋, 姚刚, 等. 牛布鲁氏菌A19突变株的构建及在BALB/c鼠中的免疫保护评估[J]. 微生物学报, 2013, 53(11): 1213-1220.
	YI Xinping, YE Feng, YAO Gang, et al. Construction of Brucella abortus A19-Δ VirB12 mutant and evaluation of its protective efficacy against 2308 strain challenge in BALB/c mice[J]. Acta Microbiologica Sinica, 2013, 53(11): 1213-1220. PMID

编辑推荐 0

Metrics

阅读次数

全文

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	7	0	0	23

来源	本网站	其他网站

次数	17	13
比例	57%	43%

摘要

121

最新录用	在线预览	正式出版

0	0	121

来源	本网站	其他网站

次数	49	72
比例	40%	60%

牛羊布鲁氏菌天然半抗原琼脂扩散试验的建立与应用

Establishment and application of natural hapten agar diffusion test for brucella

在线阅读

PDF下载

可视化

摘要/Abstract

引用本文

使用本文

图/表 10

参考文献 21

相关文章 15

编辑推荐 0

Metrics

[1]	杨川, 张凯, 陈冰, 张慧, 柳萍, 常松, 盛建东. 棉花植株形态特征对不同水分状况的响应[J]. 新疆农业科学, 2023, 60(9): 2120-2127.
[2]	金肖叶, 叶锋, 刘丽娅, 马晓菁, 李鑫, 舒展, 陈卓, 钟旗. 一起放牧羊群布鲁氏菌病的分析检测[J]. 新疆农业科学, 2023, 60(2): 479-484.
[3]	刘丽娅, 叶锋, 马晓菁, 谷文喜, 谢彩云, 钟旗, 易新萍. 布鲁氏菌病不同初筛方法及诊断试剂检测差异性分析[J]. 新疆农业科学, 2022, 59(1): 215-222.
[4]	谭京红, 吴启侠, 朱建强, 佘子浩, 柯鑫瑶, 马泓雨. 麦后移栽棉氮素营养诊断与追肥模型[J]. 新疆农业科学, 2021, 58(7): 1225-1235.
[5]	郑文强, 岳春芳, 曹伟. 基于冠气温差的阿克苏成龄红枣缺水诊断及预测[J]. 新疆农业科学, 2019, 56(12): 2256-2262.
[6]	张路瑶, 李飞, 焦海宏, 赵丽, 刘永宏. PPRV中国新疆南疆株F基因遗传演化分析[J]. 新疆农业科学, 2017, 54(11): 2118-2129.
[7]	叶静;潘存德;王世伟;肖冰;常志帅;努斯热提·托合提. 新疆乌什县新温185号核桃树体营养盈亏分析[J]. , 2014, 51(4): 614-619.
[8]	海尼木古力·艾合买提;麦丽克热·吾斯曼;林涛;胡加合买提·克依木;巴音查汗. 牛环形泰勒虫病的PCR诊断[J]. , 2014, 51(11): 2123-2126.
[9]	沙塔尔·卡哈尔;曹雯丽;张扬;王冰洁;巴音查汗. 吐鲁番部分疫区牛环形泰勒虫病流行病学调查[J]. , 2013, 50(6): 1161-1164.
[10]	蒲胜海;冯广平;李磐;张升;孙晓军;丁峰. 无土栽培基质理化性状测定方法及其应用研究[J]. , 2012, 49(2): 267-272.
[11]	黄新;康立超;韩猛立;何延华;钟发刚;薄新文. 疑似断奶仔猪多系统衰竭综合征的诊断报告[J]. , 2011, 48(9): 1747-1751.
[12]	杨阳;潘存德;王振锡;王世伟;薄翠萍;高淑然;梁燕. 轮台白杏树体营养盈亏分析[J]. , 2011, 48(4): 646-650.
[13]	张云峰;刘福元;徐雪萍;王光雷. 猪弓形虫特异PCR诊断方法的建立[J]. , 2010, 47(9): 1823-1827.
[14]	杨仁碧. 超高产棉花的产量结构及栽培技术初报[J]. , 2008, 45(z1): 49-53.
[15]	宫平;黄锡霞;田可川;徐新明;张艳花. 绵羊疫病计算机辅助诊断系统[J]. , 2008, 45(6): 1176-1181.