大鼠细小病毒H-1株和KRV株双重PCR检测方法的建立及应用

Development and application of duplex PCR for detection of H-1 and KRV strains

目的建立大鼠细小病毒H-1和KRV株双重PCR方法并对方法进行初步应用。方法根据NCBI发表的H-1(NC_001358)和KRV(U790330)基因组序列分别设计特异引物,以H-1和KRV病毒DNA为模板建立双重PCR方法,对方法进行优化后验证方法的敏感性和特异性;经口感染大鼠,分为H-1和KRV单独感染组和混合感染组,感染后第2,4,6,8,10天采集大鼠粪便,第10天处死所有大鼠,采集心、肝、脾、肺、肾和盲肠内容物等组织,用建立的方法对粪便和组织样本进行检测。结果建立的双重PCR方法以H-1和KRV为模板能够分别扩增出183 bp和302 bp 2个条带,敏感性验证显示能够检测到的最低H-1量为3.8 pg/m L,最低KRV量为0.73 pg/m L;在以小鼠微小病毒、犬细小病毒和猫细小病毒为模板时无任何条带扩增出,特异性良好。感染第2天在所有大鼠粪便中均检测到病毒核酸,感染大鼠均无明显临床症状,感染第10天采集组织,H-1在各组织的检出率分别是心50%(4/8),肝50%(4/8),脾62.5%(5/8),肺50%(4/8),肾37.5%(3/8),盲肠内容物62.5%(5/8),且单独感染组检出率高于混合感染组;KRV在各组织的检出率分别是心0(0/8),肝25%(2/8),脾87.5%(7/8),肺12.5%(1/8),肾25%(2/8),盲肠内容物62.5%(5/8),混合感染组检出率高于单独感染组。结论建立的H-1和KRV双重PCR方法能够高效的检测大鼠粪便及组织中的病毒感染,可作为实验动物国家标准的有力补充。

Objective To develop a duplex PCR assay for detection of rat parvovirus H-1 and KRV and its application. Methods To design specific primers on the basis of H-1( NC_ 001358) and KRV( U790330) genome sequences published in NCBI and establish a duplex PCR assay using H-1 and KRV DNA as templates. To verify the sensitivity and specificity of the method after optimizing PCR. The rats were infected by oral inoculation. The rats were divided into three groups: H-1 infection,KRV infection and mixed infection groups. To collect feces at the 4th,6th,8th and 10 th days postinfection. Rats were euthanized on the 10 th day and samples from heart,liver,spleen,lung,kidney and cecal contents were collected from each rat,then all the samples were screened with the duplex PCR. Results The 183 bp and 302 bp bands were amplified using H-1 and KRV as templates. The sensitivity test showed that the PCR method can detect as low as 3. 8 pg / mL H-1 and 0. 73 pg / mL KRV. There were no bands amplified when mouse minus virus,canine parvovirus and feline parvovirus were used as templates,showing that the specificity of the duplex PCR assay is very good.The nucleic acids of H-1 or KRV were detected in all rat feces on the 2th day postinfection and there was no obvious clinical symptoms in all the infected rats. The positive rates of H-1 were as follows: 50%( 4 /8) heart tissues,50%( 4 /8) liver tissues,62. 5%( 5 /8) spleen tissues,50%( 4 /8) lung tissues,37. 5%( 3 /8) kidney tissues and 62. 5%( 5 /8) cecum contents,and the positive rate of single infection group was higher than that of mixed infection group. The positive rates of KRV were as follows: 0( 0 /8) heart tissues,25%( 2 /8) liver tissues,87. 5%( 7 /8) spleen tissues,12. 5%( 1 /8)lung tissues,25%( 2 /8) kidney tissues and 62. 5%( 5 /8) cecum contents,and the positive rate of mixed infection group was higher than that of single infection group. Conclusions The duplex PCR assay for H-1 and KRV established in this study can effectively detect H-1 or KRV infection in rat feces and other tissues,and can be used as an effective supplement to the national standard of lab animals.