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1 1292 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 孟丽等 / 猪 7 种腹泻相关病毒的临床检测及猪嵴病毒的遗传进化分析 August 25, 2017, 33(8): Chin J Biotech, All rights reserved 新方法 7 孟丽 1,2, 陶洁 1, 李本强 1, 马玉飞 1,2, 程靖华 1, 张春玲 1, 刘惠莉 ,,,. 7., 2017, 33(8): Meng L, Tao J, Li BQ, et al. Clinical detection of seven porcine diarrhea-associated viruses and evolution analysis of porcine kobuvirus. Chin J Biotech, 2017, 33(8): 摘要 : 为了解引起猪腹泻疫病的病毒性病原种类及感染现状, 本研究首先针对 7 种报道较少但可引起猪腹泻的病毒性病原建立了多重 RT-PCR 检测方法, 包括猪捷申病毒 (PTV) 猪萨佩罗病毒 (PSV) 猪丁型冠状病毒 (PDCoV) 猪嵴病毒 (PKV) 猪札幌病毒 (PSaV) 猪星状病毒 (PAstV) 和猪环曲病毒 (PToV) 利用该方法对 419 份临床腹泻粪便样品进行筛检, 结果显示 PKV 检出率最高, 阳性率达 26.98% 45.79%;PKV 和其他病原混合感染率达 9.52% 18.54% 基于 PKV 的高检出率及其可能在猪腹泻疾病中发挥的作用, 本试验进一步选取 3 个 PKV 阳性样品进行全基因组序列测定及遗传进化分析结果表明, 这 3 个阳性样品 PKV 均归属于猪嵴病毒属, 且与其他动物嵴病毒遗传距离较远, 分别标记为 CM-PKV JS-PKV 和 PD-PKV; 它们的全基因组同源性为 88.1% 89.1%;CM-PKV 与 2013 年报道的中国株 JS-02a-CHN/2013 同源性最高, 而 JS-PKV 和 PD-PKV 则与 2008 年报道的匈牙利株 K-30-HUN/2008/HUN 同源性最高表明上海不同地区猪群中存在的 PKV 有明显的遗传差异, 但毒株遗传特性的差异与其致病力的相关性需进一步研究本研究为深入了解 PKV 的流行现状及探讨其在猪腹泻疫情中的作用提供了参考 : 多重 RT-PCR, 猪嵴病毒, 全基因组序列, 遗传进化分析 Received: March 24, 2017; Accepted: May 10, 2017 Supported by: Shanghai Agricultural Committee Key Project (Nos , ), Pig Industry Project of Shanghai Municipal (No ). Corresponding author: Huili Liu. Tel: ; huilil@163.com (Nos ) (No )

2 孟丽等 / 猪 7 种腹泻相关病毒的临床检测及猪嵴病毒的遗传进化分析 1293 Clinical detection of seven porcine diarrhea-associated viruses and evolution analysis of porcine kobuvirus Li Meng 1,2, Jie Tao 1, Benqiang Li 1, Yufei Ma 1,2, Jinghua Cheng 1, Chunling Zhang 1, and Huili Liu 1 1 Shanghai Engineering Research Center for Breeding Pigs, Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences, Shanghai , China 2 College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai , China Abstract: In order to analyze the epidemic situation of porcine viral diarrhea-associated diseases, a multiplex RT-PCR method was developed for detection of Seven diarrhea-associated viruses, including porcine teschovirus (PTV), porcine sapelovirus (PSV), porcine deltacoronavirus (PDCOV), porcine kobuvirus (PKV), porcine sapovirus (POSAV), porcine astrovirus (PASTV) and porcine torovirus (PTOV). A total of 419 samples were screened using the method, the results showed that PKV has the highest positive rate of 26.98% 45.79% and the mixed infection rate of PKV with others viruses were 9.52% 18.54%. On account of high positive rate of PKV and its potential important role in diarrhea disease, complete genome sequences of three PKV positive samples were further sequenced and genetic evolution were established. Sequences analysis indicated that three PKV-positive samples, designated as PD-PKV, JS-PKV and CM-PKV, respectively, were classified into the porcine kobuvirus genus. The genomic homologies of three viruses were 88.1% 89.1%. CM-PKV had the highest homology with a kobuvirus JS-02a-CHN/2013 reported in 2013, while JS-PKV and PD-PKV were most closed to a kobuvirusv K-30-HUN/2008/HUN reported in Hungary in This illustrated the significant genetic differences were existed among the PKV isolates in Shanghai. The relationship between the difference among the PKV isolates and the viral pathogenicity still needs to be explored. This research provides references for understanding the prevalence of PKV and its role in swine diarrhea disease. Keywords: multiplex RT-PCR, porcine kobuvirus, complete genomic sequences, evolution analysis (Porcine epidemic diarrhea virus, PEDV) (Transmissible gastroenteritis virus, TGEV) (Porcine rotavirus, PoRV) [1] [2] (Porcine sapelovirus, PSV) 17.2% [3] (Porcine bocavirus, PBoV) 73.95% [4] RT-PCR (Porcine deltacoronavirus, PDCoV) 31.33% Xiao [5] (Porcine astrovirus, PAstV) 11.5% (Porcine kobuvirus, PKV) TGEV [6] cjb@im.ac.cn

3 1294 ISSN CN /Q Chin J Biotech August 25, 2017 Vol.33 No.8 RNA 2007 PKV 3 [7] PKV [8-13] PKV PKV [14] 2009 PKV PKV PKV [15] PKV 7 RT-PCR PKV PKV PKV 1 材料与方法 1.1 材料 PEDV TGEV (Classical swine fever virus, CSFV) (Bovine viral diarrhea virus, BVDV) (Swine influenza virus, SIV) bp DNA ladder marker pmd-18 T rtaq DNA TRIzol M-MLV (RNase H-) dntps TaKaRa DNA Tiangen DH5α PCR (ABI) (Beckman) (Vision) (Bio-Rad) Q 方法 [2-6] GenBank PKV (EU KT JX KM977675) PAstV (JX KU JX JX556691) PSaV (KT FJ KT KF204570) PDCoV (KJ KR KT KX022605) PToV (GU JQ FJ555569) PTV (AF AF GU JQ AJ516011) PSV (HQ KJ KJ821021) Oligo7.37 PTV 3D PKV 3D PDCoV M PSV 5 UTR PSaV ORF1 PAstV ORF2 PToV S 7 ( 1) ( ) RNA PBS r/min 2 min 250 μl RNA cdna

4 孟丽等 / 猪 7 种腹泻相关病毒的临床检测及猪嵴病毒的遗传进化分析 1295 表 1 猪 7 种病毒性腹泻病原的多重 PCR 引物序列 Table 1 Multiplex PCR primers of the seven porcine diarrhea-associated viruses Name Primer sequences (5 3 ) Product length (bp)* Location (nt)* PTV-F ATGGGACTCTAGATCTCGT PTV-R ACGCCTCTGTAGTTCTCTCTT PAstV-F GGATTTACAGTTGGCCCAGAT PAstV-R CCTGTCCATCTGCCTTTCTGT PKV-F ATGCTGCTTGGTGGACTCATT PKV-R ACGTCTCGTTGCCAAAGACAT P-SaV-F CGAGGCTAAAGGGAAAAACAAACGT P-SaV-R ACTGTCGTAGGTGTCTGCTTTT PToV-F CTTTTACACCTTGCCATCC PToV-R TGCTTCACCTCTACACTGTT PDCoV-F ACTTATTCTGCTTTGGCTGCT PDCoV-R GAAGTGGTTATGGTGTGAAGTC PSV-F GATGTGGCGCATGCTCTT PSV-R TGCTGCCTCCTGTGTTGTTAT *Primer locations and lengths are listed according to the strain PTV (AF231767), PAstV (JX556692), PKV (EU787450), PSaV (KT922089), PToV (GU196786), PDCoV (KJ769231) and PSV (HQ875059), respectively RT-PCR cdna RT-PCR 7 RT-PCR RT-PCR RT-PCR [16] 8 PKV 3 3 PKV 5 5 RACE kit 3 ( ) Lasergene PKV MEGA6.0 2 结果与分析 2.1 阳性质粒的克隆和鉴定 7 ( 1) peasy-t1 Simple Cloning Kit TA DH5α PCR peasy-ptv/3d peasy-pkv/3d peasy-pdcov/m peasy- PSV/5 UTR peasy-psav/orf1 peasy-pastv/ ORF2 peasy-ptov/s cjb@im.ac.cn

1296 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech August 25, 2017 Vol.33 No.

1 Specific fragments amplification of the seven porcine diarrhea-associated viruses.

fragment; 5: PToV S fragment; 6: PDCoV M fragment; 7: PSV 5 UTR fragment. 2.

1 RT-PCR PEDV TGEV CSFV BVDV SIV 4 PTV PKV PDCoV PSV PSaV PAstV PToV RT-PCR 7 ( 4) 图 2 多重 RT-PCR

M: 100 bp DNA marker; 1: multiplex RT-PCR for PTV and PSV; 2: multiplex PCR for PTV, PDCoV and

5 1296 ISSN CN /Q Chin J Biotech August 25, 2017 Vol.33 No.8 图 1 猪 7 种病毒性腹泻病原的目的片段扩增 Fig. 1 Specific fragments amplification of the seven porcine diarrhea-associated viruses. M: 100 bp DNA marker; 1: PTV 3D fragment; 2: PAstV ORF2 fragment; 3: PKV 3D fragment; 4: PSaV ORF1 fragment; 5: PToV S fragment; 6: PDCoV M fragment; 7: PSV 5 UTR fragment. 2.2 多重 PCR 的建立 7 RT-PCR 7 RT-PCR ( 2) bp 504 bp 421 bp 340 bp 280 bp 249 bp 176 bp PCR 58 PCR 2.3 多重 RT-PCR 特异性和灵敏性的鉴定 RT-PCR PEDV TGEV CSFV BVDV SIV 4 PTV PKV PDCoV PSV PSaV PAstV PToV RT-PCR 7 ( 4) 图 2 多重 RT-PCR 的建立 Fig. 2 Development of the multiplex RT-PCR. M: 100 bp DNA marker; 1: multiplex RT-PCR for PTV and PSV; 2: multiplex PCR for PTV, PDCoV and PSV; 3: multiplex RT-PCR for PTV, PToV, PDCoV and PSV; 4: multiplex RT-PCR for PTV, PSaV, PToV, PDCoV and PSV; 5: multiplex RT-PCR for PTV, PKV, PSaV, PToV, PDCoV and PSV; 6: multiplex RT-PCR for PTV, PAstV, PKV, PSaV, PToV, PDCoV and PSV; M: marker. 图 3 多重 RT-PCR 的退火温度筛选 Fig. 3 Temperature screening of the multiplex RT-PCR. 1 4: T m 52, 55, 58 and 61 ; M: marker PCR 10 5 PCR PCR PTV PKV

孟丽等 / 猪 7 种腹泻相关病毒的临床检测及猪嵴病毒的遗传进化分析 1297 PDCoV PSV PSaV PAstV PToV 5.0 10 4 5.5 10 4 6.0 10 4 6.0 10 4 3.8 10 4 6.1 10 4 6.9 10 4 copies/μl 0.32 0.36 0.41 0.41 0.18 0.39 0.43 μg/l ( 4) 2.3.3 RT-PCR 5 2.

4 Specificity and sensitivity of the multiplex RT-PCR.

6 孟丽等 / 猪 7 种腹泻相关病毒的临床检测及猪嵴病毒的遗传进化分析 1297 PDCoV PSV PSaV PAstV PToV copies/μl μg/l ( 4) RT-PCR 多重 RT-PCR 与单项 RT-PCR 临床检测结果的比较 RT-PCR PKV % 45.79% 2 PKV 9.52% (2015 ) 18.54% (2016 ) RT-PCR 7 100% PKV 全基因组序列的测定 PKV 3 PKV 5 图 4 多重 RT-PCR 特异性和灵敏性鉴定 Fig. 4 Specificity and sensitivity of the multiplex RT-PCR. M: 100 bp DNA ladder; 1: multiplex RT-PCR used PTV, PKV, PDCoV, PSV, PSaV, PAstV and PToV as the templates; 2: multiplex RT-PCR used PEDV as the template; 3: multiplex RT-PCR used TGEV as the template; 4: multiplex RT-PCR used BVDV as the template; 5: multiplex RT-PCR used CSFV as the template; 6: multiplex RT-PCR used SIV as the template; 7 12: sensitivity detection of the multiplex RT-PCR with the plasmids of Lasergene Cluster X 3 PKV PD-PKV JS-PKV CM-PKV PD-PKV JS-PKV CM-PKV bp bp bp 5 UTR 3 UTR ORF PD-PKV JS-PKV aa CM-PKV aa 表年猪病毒性腹泻病原的阳性检出率 Table 2 Infection rate of the porcine diarrhea-associated viruses during 2015 to 2016 Year Total samples Infection rate (%) PTV PToV PDCoV PKV PSaV PAstV PSV Coinfection rate of PKV and other viruses cjb@im.ac.cn

1298 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech August 25, 2017 Vol.33 No.8 2.6 全基因组遗传进化分析 GenBank MEGA6.0 3 ( 6) JS-PKV PD-PKV JS-PKV JS-2010a 图 5 PKV 全基因组 PCR 扩增 Fig.

PDPKV K-4/2012/CH JS-01-CHN/2013/China 表 3 全基因组进化树参考毒株 Table 3 Reference strains of the phylogenetic tree based on the complete genomic sequences Strains Origins GenBank No.

7 1298 ISSN CN /Q Chin J Biotech August 25, 2017 Vol.33 No 全基因组遗传进化分析 GenBank MEGA6.0 3 ( 6) JS-PKV PD-PKV JS-PKV JS-2010a 图 5 PKV 全基因组 PCR 扩增 Fig. 5 PCR amplification of the complete genomic sequence of PKV. PDPKV K-4/2012/CH JS-01-CHN/2013/China 表 3 全基因组进化树参考毒株 Table 3 Reference strains of the phylogenetic tree based on the complete genomic sequences Strains Origins GenBank No. Aichi virus Chshc7 China FJ (2008) Aichi virus kvgh /2010 Taiwan, China JX (2010) Aichi virus D/VI2359/2004 Germany GQ (2004) Aichi virus Goiania/GO/03/01/Brazil Germany DQ (2006) Bovine kobuvirus U-1 Japan AB (2008) Bovine kobuvirus SC1 Scotland KT (2013) Mouse kobuvirus M-5/USA/2010 USA JF (2010) Canine kobuvirus UK003 UK KC (2008) Canine kobuvirus CH-1 Hubei, China JQ (2011) Canine kobuvirus US-PC0082 USA JN (2010) Canine kobuvirus B103 Germany KM (2010) Canine kobuvirus AN211D/USA/2009 USA JN (2009) Feline kobuvirus FeKoV/TE/52/IT/13 Italy KM (2013) Feline kobuvirus FK-13 Korea KF (2011) Porcine kobuvirus JS-02a-CHN/2013 Jiangsu, China KP (2013) Porcine kobuvirus JS-01-CHN/2013 Jiangsu, China KP (2013) Porcine kobuvirus K-4/2012/CH Gansu, China KC (2012) Porcine kobuvirus JY-2010a Beijing, China GU (2010) Porcine kobuvirus ch-kobu/2008/china Jiangsu, China KF (2008) Porcine kobuvirus WB-1-HUN/2011/HUN Hungary JX (2011) Porcine kobuvirus K-30- HUN/2008/HUN Hungary GQ (2008) Caprine kobuvirus 12Q108 South Korea KF (2012) Sheep kobuvirus TB3/HUN/2009 Hungary GU (2009)

8 孟丽等 / 猪 7 种腹泻相关病毒的临床检测及猪嵴病毒的遗传进化分析 1299 图 6 PKV 全基因组进化树分析 Fig. 6 Phylogenetic tree based on the complete genomic sequences of PKV. 3 PKV 88.1% 89.1% ( 7) CM-PKV JS-02a- CHN/2013/China (89.5%) JS-PKV PD-PKV K-30-KUN/2008/HUN (89.6%) 2.7 VP1 基因的遗传进化分析 VP1 PKV VP1 ( 8) JS-PKV PD-PKV CM-PKV JS-PKV CM-PKV 2007 CMP03/07 CMP17/07-THA PD-PKV 2009 T247/2009/JP 3 PKV cjb@im.ac.cn

1300 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech August 25, 2017 Vol.33 No.8 图 7 PKV 全基因组序列的同源性比对 Fig.

9 1300 ISSN CN /Q Chin J Biotech August 25, 2017 Vol.33 No.8 图 7 PKV 全基因组序列的同源性比对 Fig. 7 Identity analyzation of the complete genomic sequences of PKV. 图 8 VP1 基因的进化树分析 Fig. 8 Phylogenetic tree based on the VP1 genes.

10 孟丽等 / 猪 7 种腹泻相关病毒的临床检测及猪嵴病毒的遗传进化分析讨论 PEDV TGEV PoRV RT-PCR [1,17-21] 3 RT-PCR 4 RT-PCR 7 RT-PCR PTV PKV PDCoV PSV PSaV PAstV PToV μg/l RT-PCR [22-26] PKV 180 PTV PDCoV 25 PToV 10 PAstV 108 PSV 75 PKV 72 PCR 7 RT-PCR PKV 3 PKV JS-PKV PD-PKV CM-PKV PKV 5 UTR ORF 3 UTR polya bp bp bp G+C 52.07% 52.04% 52.09% A+T 47.93% 47.96% 47.91% 88.1% 89.1% PKV JS-PKV PD-PKV K-30-KUN/2008/HUN 89.6% CM-PKV JS-02a-CHN/2013/China 89.5% 3 PKV VP1 PKV PKV PKV 3 PKV REFERENCES [1] Ogawa H, Taira O, Hirai T, et al. Multiplex PCR and multiplex RT-PCR inclusive detection of major swine DNA and RNA viruses in pigs with multiplex infections. J Virol Methods, 2009, 160(1/2): [2] Lan DL, Ji WH, Wang CS, et al. Molecular epidemiology investigation of porcine sapelovirus in pigs in East China. Prog Vet Med, 2012, 33(12): (in Chinese).,,,.., 2012, 33(12): [3] Cai YH, Zhu L, Zhou YC, et al. Development and application of the duplex PCR for detection of porcine bocavirus. Chin Vet Sci, 2013, 43(8): (in Chinese).,,,. PCR., 2013, 43(8): [4] Liu LL, Cao BB, Zhang LW, et al. Establishment and preliminary application of duplex RT-PCR for detection of emerging porcine deltacoronavirus and porcine transmissible gastroenteritis virus. J Agricul Biotechnol, 2016, 24(12): (in Chinese).,,,. PDCoV TGEV RT-PCR., 2016, 24(12): cjb@im.ac.cn

11 1302 ISSN CN /Q Chin J Biotech August 25, 2017 Vol.33 No.8 [5] Xiao CT, Luo Z, Lv SL, et al. Identification and characterization of multiple porcine astrovirus genotypes in Hunan province, China. Arch Virol, 2017, 162(4): [6] Jackova A, Sliz I, Mandelik R, et al. Porcine kobuvirus 1 in healthy and diarrheic pigs: Genetic detection and characterization of virus and co-infection with rotavirus A. Infect Genet Evol, 2017, 49: [7] Jin WJ, Yang Z, Zhao ZP, et al. Genetic characterization of porcine kobuvirus variants identified from heathy pigs in China. Infect Genet Evol, 2015, 35: [8] Yu JM, Jin M, Zhang Q, et al. Candidate porcine kobuvirus, China. Emerg Infect Dis, 2009, 15(5): [9] Wang C, Lan D, Hua X. Porcine kobuvirus from pig stool specimens in Shanghai, China. Virus Genes, 2011, 43(3): [10] Khamrin P, Maneekarn N, Kongkaew A, et al. Porcine kobuvirus in piglets, Thailand. Emerg Infect Dis, 2009, 15(12): [11] Park SJ, Kim HK, Moon HJ, et al. Molecular detection of porcine kobuvirus in pigs in Korea and their association with diarrhea. Arch Virol, 2010, 155(11): [12] Khamrin P, Maneekarn N, Hidaka S, et al. Molecular detection of kobuvirus sequences in stool samples collected from heathy pigs in Japan. Infect Genet Evol, 2010, 10(7): [13] Barry AF, Ribeiro J, Alfieri AF, et al. First detection of kobuvirus in farm animals in Brazil and the Netherlands. Infect Genet Evol, 2011, 11(7): [14] Zhao ZP, Yang Z, Lin WD, et al. The rate of co-infection for piglet diarrhea viruses in China and the genetic characterization of porcine epidemic diarrhea virus and porcine kobuvirus. Acta Virol, 2016, 60(1): [15] Phan NT, Trinh QD, Khamrin P, et al. Sequence analysis of the capsid gene of Aichi viruses detected from Japan, Bangladesh, Thailand, and Vietnam. J Medical Virol, 2008, 80(7): [16] Peng Q, Song D, Huang D, et al. Complete genome sequence of a porcine kobuvirus variant strain from Jiangxi, China. Genome Announc, 2017, 5(5): pii: e [17] Ben Salem AN, Chupin Sergei A, Bjadovskaya Olga P, et al. Multiplex nested RT-PCR for the detection of porcine enteric viruses. J Virol Methods, 2010, 165(2): [18] Zhao J, Shi BJ, Huang XG, et al. A multiplex RT-PCR assay for rapid and differential diagnosis of four porcine diarrhea associated viruses in field samples from pig farms in East China from 2010 to J Virol Methods, 2013, 194(1/2): [19] Song DS, Kang BK, Oh JS, et al. Multiplex reverse transcription-pcr for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group A rotavirus. J Vet Diagn Invest, 2006, 18(3): [20] Ren YP, Zhang B, Tang C, et al. Development and application of a multiplex RT-PCR for detecting PEDV, TGEV and PDCoV. Chin Vet Sci, 2016, 46(6): (in Chinese).,,,. PEDV TGEV PDCoV RT-PCR., 2016, 46(6): [21] Xiong NN, Tao J, Zhang QZ, et al. Establishment and application of the multiplex PCR for detection of four porcine viral diarrhea viruses. Chin J Vet Sci, 2016, 46(8): (in Chinese).,,,. PCR., 2016, 46(8): [22] Li LM, Zhu WX, Wang XB, et al. Establishment and application of a multiplex reverse transcription-pcr for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine group A rotavirus. Chin J Vet Sci, 2016, 36(2): (in Chinese).,,,. A

12 孟丽等 / 猪 7 种腹泻相关病毒的临床检测及猪嵴病毒的遗传进化分析 1303 R-PCR., 2016, 36(2): [23] Long FX, Shi KC, Zhang Z, et al. Establishment and application of a multiplex RT-PCR assay for detection of PEDV, TGEV and PRoV. Chin Anim Husb & Vet Med, 2016, 43(10): (in Chinese).,,,. PEDV TGEV PRoV RT-PCR., 2016, 43(10): [24] Yang WY, Zhou YC, Han Y, et al. Establishment and clinical application of a multiplex reverse transcription-pcr for simultaneous detection of porcine group A rotavirus, porcine group C rotavirus and porcine astrovirus. Chin Vet Sci, 2014, 44(4): (in Chinese).,,,. A C RT-PCR., 2014, 44(4): [25] Gu F, Zhou YC, Huang JB, et al. Establishment and clinical application of a multiplex RT-PCR for detection of porcine kobuvirus, porcine astrovirus and porcine torovirus. Chin Vet Sci, 2015, 45(7): (in Chinese).,,,. RT-PCR., 2015, 45(7): [26] Xu YD, Zhang QX, Li JL, et al. Establishment and application of multiple RT-PCR method for diagnosis of PEDV, TGEV and RV infection. Chin J Vet Drug, 2014, 48(11):10 13 (in Chinese).,,,. PEDV TGEV RV RT-PCR., 2014, 48(11): ( 本文责编陈宏宇 ) cjb@im.ac.cn

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr PCR 龙平, 崔占虎, 李虔全, 徐建平, 张春红, 周立社 * *, 李旻辉 ( 包头医学院, 内蒙古包头 014060) DNA 30 28 DNA trnl-trnf PCR PCR 136 bp 323 bp 10% PCR PCR Astragalus membranaceus Fisch. Bge. var. mongho- licus Bge. Hsiao A. membranaceus