广西农垦永新畜牧集团良圻原种猪场

PRRS Detection Accuracy Using Different Primers

用不同引物检测PRRS的准确性

Liangqi Farm of Yongxin Husbandry Group Co., Ltd of Guangxi State Farms Wu shaoqin 伍少钦

wsqin326@163.com

广西农垦永新畜牧集团良圻原种猪场

Background/背景资料

Ø A 1200- sow customer farms, finish pigs found sick in December,某客户猪场，规模1200头母猪，育肥猪场于12月有问题出现：

Ø No. 2 finish barn with 406 pigs; Dec 13, pigs found to have fever, abdominal breathing, panting, dark blue ear tip in late stage, bloody foam from nose after death, weight 90-95kg;育肥第2栋，共406头，12月13日开始发现有猪发烧，体温升高，腹式呼吸、喘气、后期耳尖呈暗蓝色，死亡后鼻孔流出带血泡沫。平均体重在90-95kg；

Ø Dec.14, # sick pigs increased, 28 new cases, 2 died, mostly in the 5 to 8 pens. Pigs injected with florfenicol, which was added in water at 50g/ T; after 6-day treatment, disinfection with pigs.14日第2栋发现发病猪增多，发病28头，死亡2头。发病主要集中在前面5至8栏。病猪用氟苯尼考肌注，同时水中加氟苯尼考50g/T，连用6天，带猪消毒

Ø Death :1/Dec.15, 1/Dec.16, 3/Dec.17, 15号死亡1头，16号死亡1头，17号死亡3头。

广西农垦永新畜牧集团良圻原种猪场

Ø Dec. 18 ,veterinarian on site autopsy 2 pigs , Clinically suspected Actinobacillus pleuropneumoniae (APP), 1 death.12月18号猪场兽医现场查看猪群，并解剖两头。初步诊断为传染性胸膜肺炎。死亡1头

Ø Dec. 19 , in feed added: 2%Nuflor 2Kg/T + 15%CTC 2Kg/T , 7 days, 6 deaths.12月19日料加纽弗罗2Kg/T+金霉素2Kg/T,7天，死亡6头

Ø Dec. 20, whole herd injection with ceftiofur sodium, 450mg/head,3 days, 5 deaths.12月20号全群普防头孢噻呋钠450mg/头连打3天，死亡5头。

Ø Then herd remained stable for 10 days, 3 deaths . 猪群稳定有10天左右，共死亡3头。

Ø Dec. 31 morning, sudden death of 3 pigs , another 3 pigs got sick. dead pigs had bloody foam in the mouth and nasal, suspected of APP. 7 death in the same day. whole herd injection with ceftiofur sodium 450mg/head for 5 days, 12月31号早上死亡急性3头，还有3头在发病。死亡后口鼻腔流泡沫血，疑似传染性胸膜肺炎。全天死亡7头。病猪集中在下风口1-3栏。全群普防头孢噻呋钠450mg/头连打5天；

广西农垦永新畜牧集团良圻原种猪场

临床上疑似APP

Clinically suspected Actinobacillus

pleuropneumoniae(APP)

广西农垦永新畜牧集团良圻原种猪场

Vet lab test 实验室检测

Ø Clinically, was diagnosed as common bacterial disease(APP), with no sufficient attention paid. Treatment showed some effects but did not effectively control. In January, blood samples were tested again in the lab. 从临床症状，初诊为常见细菌病(APP)，当时没有引起足够重视，用药后有一定效果，但没得到有效控制，所以1月初，要求重新采血送检；

Ø Jan. 2, bleed the sick pig, Jan. 5 vet lab ran test on PRRS antibody, results as follows: 1月2号对发病猪采血送检，兽医实验室1月5日检测PRRS抗体结果如下：

广西农垦永新畜牧集团良圻原种猪场

IDEXX PRRS X3 Ab Test

No. PRRS OD S/P value comment Test time

1 1.178 2.486 APP (?)pig 20130105

2 0.942 1.96 APP (?)pig 20130105

3 0.728 1.483 APP (?)pig 20130105

4 0.324 0.584 APP (?)pig 20130105

5 0.418 0.793 APP (?)pig 20130105

6 0.719 1.463 APP (?)pig 20130105

7 0.641 1.29 APP (?)pig 20130105

8 0.696 1.412 APP (?)pig 20130105

9 1.024 2.143 APP (?)pig 20130105

10 0.706 1.434 APP (?)pig 20130105

广西农垦永新畜牧集团良圻原种猪场

Sera RT-PCR 重新采集血清 RT-PCRNO. PRRSV NO. PRRSV

1 - 13 -2 - 14 -3 - 15 -4 - 16 -5 - 17 -6 - 18 -7 - 19 -8 - 20 -9 - 21 -

10 - 22 -11 - 23 -12 - Positive control +

广西农垦永新畜牧集团良圻原种猪场

用ORF5引物（773bp）无法从以下样品血清中扩增获得目的片段。

Ø Using ORF5 primer, the result is negative

广西农垦永新畜牧集团良圻原种猪场

20130121检测结果 20130125检测结果

编号 耳号 PRRS-ORF5 编号 耳号 PRRS-ORF7

1 L44502 1 L44502

2 264704 2 264704

3 L283701 3 L283701

4 L245304 4 L245304

5 L283508 5 L283508

6 L91005 6 L91005

7 L61508 7 L61508

8 L272701 8 L272701

9 L227001 9 L227001

10 Y281306 10 Y281306

11 Y264801 11 Y264801

12 L79203 12 L79203

13 L42701 13 L42701

14 L40002（可疑阳性） 14 L40002

15 Y244106 15 L40002

16 L18304

17 Y262302（死亡） 16 Y262302（死亡）

18 Y252602 17 Y252602

19 Y43307 18 Y43307

20 L271204 　 　

21 L266704 19 L266704

22 Y284301 20 Y284301

23 2头 21 35405、27104、19301、28901、29311

24 Positive control 22 34601、28803、35708、17503、28505

　 　 　 23 ?、29005、25201、29307、21304

　 　 　 24 Positive control (300bp)

广西农垦永新畜牧集团良圻原种猪场

Ø In Jan.25 testing, samples 1-14,17-19, 21, and 22 ,are for retesting corresponding samples run on Jan 21using ORF7 primer(300bp). After using ORF7 primer（300bp) on Jan 25,positive increase to 68.4%, while Jan 21 test using ORF5 were all negative.

Ø 20130125检测样品中，编号1-14、17-19、21、22为利用PRRS-ORF7引物(300bp)复检20130121相应编号的样品。20130121（ORF5)全为阴性20130125用ORF7引物复检阳性率达68.4%。

广西农垦永新畜牧集团良圻原种猪场

Ø把猪场育成猪PRRS抗体呈阳性的血清，进一步用ORF7引物进行检测，发现 S/P值的高低与抗原的检出率没有什么相关性，监测数据如下表：

Ø Collected PRRS antibody positive sera from finish pigs, and then tested using ORF7 primers, and found there is no correlation between S/P value and the PRRSV antibody detection. Data as follows:

广西农垦永新畜牧集团良圻原种猪场

No. type PRRS-S/P value RT-PCR (ORF7 300bp)

1 finish pig 0.207 “+”

2 finish pig 0.22 “+”3 finish pig 0.227 “+”4 finish pig 0.248 “+”5 finish pig 0.258 “+”6 finish pig 0.289 “+”7 finish pig 0.405 “+”8 finish pig 0.433 弱“+”9 finish pig 0.435 “+”10 finish pig 0.589 “+”11 finish pig 0.756 弱“+”12 finish pig 0.903 弱“+”13 finish pig 0.962 “+”14 finish pig 1.108 弱“+”15 finish pig 1.516 “+”16 finish pig 1.727 “+”17 finish pig 2.211 弱“+”

广西农垦永新畜牧集团良圻原种猪场

Sensitivity of 2 pairs PRRSV primers 两对PRRSV引物检测灵敏度对比

编号N0. 稀释 Diluted 结果result Primer 检测时间/test time

1 400μl Serum “+” ORF5 20110217

2 40μl serum+360μl water “+” ORF5 20110217

3 20μl serum+380μl water “+” ORF5 20110217

4 8μl serum+392μl water “+” ORF5 20110217

5 4μl serum+396μl water - ORF5 20110217

6 20μl serum+380μlwater “+” ORF7 20130217

7 8μl serum+392μl water “+” ORF7 20130217

8 4μl serum+396μl water “+” ORF7 20130217

9 2μl serum+398μl water “+” ORF7 20130217

10 1μl serum+399μl water “+” ORF7 20130217

样品-2011年制备的驯化血清，ORF5检测片段是5773bp，ORF7是300bp，大致说明，在同等条件下，300bp引物检测敏感性至少比773bp高8倍Sample - serum prepared for PRRS acclimation in 2011, ORF5 objective fragment is 773bp, ORF7 300bp. In general, 300bp primer sensitivity is at least 8 times higher than that of 773bp.

广西农垦永新畜牧集团良圻原种猪场

The client serum and M, were diluted as 1:15 (20μL serum in 280μL DEPC water), 1:30, 1:60, and 1:120.

The RT-PCR was performed with the serums and the diluted serums as template.

RESULT: 用ORF5引物（773bp）无法从客户血清和四种稀释度的客户血清中扩增获得目的片段；而ORF7引物（300bp）可以从客户、M血清和四种稀释度的两种血清中扩增获得目的片段。

The primer ORF5 (773bp) cannot get the amplified fragment in serum; the ORF7 primers (300bp) can detect the virus from sera ( M and client including 1:120 diluted)

原液M 原液1:15 1:30 1:60 1:120 1:15 1:30 1:60 原液 原液1:15 1:30 1:60 1:120 1:15 1:30 1:60 1:1201:120

2000bp

750bp500bp

250bp100bp

1000bp

20130811

广西农垦永新畜牧集团良圻原种猪场

M Virus 1:102 1:104 1:106 1:102 1:104 1:106Virus

ORF5 ORF7

The virus (104.75TCID50/0.1mL) was diluted to 1:102, 1:104, 1:106 in DEPC water.

以病毒及稀释过的病毒为模板，并分别以ORF5（773bp）、ORF7（300bp）引物进行RT-PCR，结果发现，以ORF5（773bp）引物可以从病毒原液中扩增获得目的片段，却不能从稀释过的病毒液中扩增到；而以ORF7（300bp）引物可以从病毒原液及稀释过的病毒液中扩增获得目的片段（从图中可见，稀释到1:102, 1:104 时，可以获得亮度相当的目的带，稀释到1:106时，可以获得亮度较弱的目的带。

2000bp

750bp500bp

250bp

100bp

1000bp

20130811

广西农垦永新畜牧集团良圻原种猪场

M 1:240 1:480 1:108

ORF5 ORF7

1:240 1:480 1:240 1:480 1:240 1:480 1:108

客户 M 客户 M Viru

s-ORF5

Viru

s-ORF7

2000bp

750bp

500bp

250bp

100bp

1000bp

20130812

当使用ORF7检测时，检测案例分析中的客户血清稀释2000倍还可检到；检测M血清时，可以检测到稀释18000倍的血清，When using ORF7 primer, virus can be detected in a customer (case study) serum at 2000 dilution; in M serum at 18000 dilution.

广西农垦永新畜牧集团良圻原种猪场

M 1:500 1:2000 1:6000 1:18000

客户1:500 1:2000 1:6000 1:18000

M1:1010 1:1012

Virus

2000bp

750bp

500bp

250bp

100bp

1000bp

20130813

当使用ORF7检测时，检测案例分析中的客户血清稀释2000倍还可检到；检测M血清时，可以检测到稀释18000倍的血清，JXA1病毒液稀释1012 ，When using ORF7 primer, virus can be detected in a customer (case study) serum at 2000 dilution; in M serum at 18000 dilution.

广西农垦永新畜牧集团良圻原种猪场

附：两对引物及反应条件Two primers and reaction conditionsØ 引物序列： ORF5-773bp primer: PRRSVF:CTG AGA CCA TGA GGT GGG CAA CT

primer PRRSVR:CAT CAC TGG CGT GTA GGT AAT AGA AAA C ORF7-300bp primer: PRRSVF:CCA GCC AGT CAA TCA RCT GTG PRRSVR: GCG AAT CAG GCG CAC WGT ATG

Ø 反应条件：

773bpF/R：95 ℃ 5 min，95 ℃ 1min，57 ℃ 1min，72 ℃ 1 min，35个循环， 72 ℃ 10 min 300bpF/R：95 ℃ 15 min，95 ℃ 30 s，60 ℃ 30 s，72 ℃ 1 min， 40个循环， 72 ℃ 10 min

广西农垦永新畜牧集团良圻原种猪场

参考文献 Reference

Ø Donadeu, M., Arias, M., Gomez-Tejedor, C., Aguero, M., Romero, L.J., Christianson, W.T., Sanchez-Vizcaino, J.M., 1999. Using polymerase chain reaction to obtain PRRSV-free piglets from epidemically infected herds. Swine Health Prod. 7,225–261.

Ø Toplak I, Rithtaric D, Hostnik J, Grom J, Stukelj M, Valencak Z. 2012.Identification of a genetically diverse sequence of porcine reproductive and respiratory syndrome virus in Slovenia and the impact on the sensitivity of four molecular tests. J. Virol. Methods 179:51–56.

广西农垦永新畜牧集团良圻原种猪场

Acknowledgements 致 谢Ø 永新畜牧集团公司吴志君总经理 general manager

Yongxin Wu Zhijun, Ø 美国谷物协会北京办闫之春技术主任 U.S. Grains Council

office in Beijing Jason YanØ 良圻原种猪场兽医实验室全体成员 All members of the

Liangqi pig farm veterinary laboratories Ø 永新良圻原种猪场全体员工 All members of Yongxin

Liangqi pig farms Ø 广西兽医研究所马玲研究生 Ms Ma Ling, Guangxi

Veterinary Research InstituteØ 广西大学动科院谢体三博士 Dr. Xie Tisan, Animal

Science College of Guangxi University Ø 海博莱曾容愚博士 Dr. Zeng Rongyu HIPRA company

广西农垦永新畜牧集团良圻原种猪场

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