目的  本研究旨在讨论脊髓灰质炎疫苗不同序贯免疫的接种效果和排毒情况，为制定合理的脊髓灰质炎疫苗免疫程序提供依据；并比较了不同的脊髓灰质炎检测方法，为选择合理的脊髓灰质炎病毒检测方法提供参考。

方法  选取广西壮族自治区1200名2~3月龄健康的未接种过脊灰疫苗的婴幼儿，随机分为IPV和OPV序贯免疫组，共12组，每组100人，在第0、28、56天接种相应疫苗，在免前和基础免疫后28天检测血清中脊灰各型抗体阳转率和抗体滴度，并随机抽取各组入组时前10%的受试对象在第二剂接种前和第二、三接种后第7、14、28天进行粪便采集，总共7次，所有的粪便样本通过L20B和RD两种细胞系进行分离培养，通过中和试验对细胞培养物中脊灰病毒进行血清型别鉴定。比较各序贯免疫程序组不同时期各型病毒的排毒率情况。第二部分从上述免疫程序组每组抽取6个粪便样本，通过实时荧光定量PCR法检测粪便上清中脊灰病毒的排毒情况，并与细胞培养法进行比较，分别用ELISA、RT-PCR对细胞培养物中的脊灰病毒进行型内鉴定，比较中和试验、ELISA、RT-PCR三种方法分型结果的一致性。

结果  排除脱落与其他不符合实验方案的受试者，总共2062份配对血清和806份粪便样本进入统计学分析。12个序贯免疫组各组免前脊灰各型抗体阳性率和GMT均无统计学差异。相同免疫程序不同剂型免后脊灰各型抗体阳转率和GMT均无显著差异。相同剂型不同免疫程序免后I、III型抗体阳转率组间比较均无显著差异，2IPV+bOPV组免后脊灰I、III型抗体GMT均高于其他组；接种2剂IPV后II型抗体阳转率和GMT显著升高。cIPV/OPV序贯免疫组与sIPV/OPV序贯免疫组相同免疫程序免后各型脊灰抗体阳转率均无统计学差异；sIPV/OPV序贯免疫组免后I型抗体GMT均高于相同免疫程序的cIPV/OPV序贯免疫组，II型抗体低于cIPV/OPV序贯免疫组，接种2剂sIPV后与cIPV/OPV序贯免疫组无显著差异；IPV/OPV序贯免疫组与sIPV/OPV序贯免疫组免后III型抗体GMT无显著差异。各免疫程序组在接种首剂OPV后7天内排毒率达到最高峰，随后排毒率逐渐降低。首剂OPV接种后3周内均出现任意一型病毒的排毒，且III型脊灰病毒排毒率最高。各免疫程序组I、II型脊灰病毒排毒率无显著差异；sIPV+2bOPV组III型病毒总排毒率显著高于2sIPV+bOPV组与2sIPV+tOPV组，cIPV+2bOPV液体组显著高于2cIPV+tOPV液体组。细胞培养法检测出的阳性样本用实时荧光定量PCR法均能检测出来，且实时荧光定量PCR法检测的阳性结果更多；中和试验、ELISA、RT-PCR三种方法对细胞培养物中脊灰病毒的分型结果无显著差异。

结论  脊灰疫苗基础免疫效果受到不同序贯免疫程序的影响，2剂IPV免疫效果优于1剂IPV，尤其是II型。不同的脊髓灰质炎序贯免疫排毒情况有差异，IPV剂次增加虽然不能显著降低排毒率，但能缩短各型病毒的排毒时间。实时荧光定量PCR法对原始粪便标本中脊灰病毒的检出率比细胞培养法更高，但无法判断病毒是否具有感染性，因此样本量较大时可以用来初步筛选出阳性样本，然后再用细胞培养法进行检测。中和试验、ELISA、RT-PCR三种方法对脊灰病毒的分型结果一致性很高，可以使用后面的两种方法替代经典的中和试验法来对细胞培养物中的脊灰病毒进行型内鉴定。

关键词 ：脊髓灰质炎灭活疫苗，脊髓灰质炎减毒活疫苗，序贯免疫，粪便排毒

ive  this study is aim to discuss the vaccination effect and detoxification of different sequential immunization of polio vaccine to provide a theoretical basis for develop a reasonable immunization procedures of polio vaccine. then we compare the different poliovirus detection methods to provide a reference for the selection of reasonable detection methods of poliovirus.

methods  a total of 1200 healthy infants aged 2 ~ 3 months without a history of polio vaccine in the guangxi zhuang autonomous region were randomly divided into ipv and opv sequential immunization groups. the seroconversion rate and antibody titer of polio antibodies in serum were detected before immunization and 28 days after basic immunization, and 10% of the subjects in each group were collected before the second dose of vaccination and on the 7th, 14th and 28th day after the second and third vaccination. a total of 7 times, all fecal samples were isolated and cultured by l20b and rd cells and the serotypes of poliovirus in cell cultures were identified by neutralization test. the detoxification rates of different types of viruses in different sequential immunization program groups were compared. in the second part, six fecal samples were taken from each group of the above immune program group, and the excretion of poliovirus in fecal supernatant was detected by real-time quantitative pcr, and compared with cell culture method. the intratype identification of poliovirus in cell culture was carried out by elisa and rt-pcr, respectively, and the results of neutralization test, elisa and rt-pcr were compared. 
results  finally a total of 2062 serum specimen and 806 fecal samples were involved in the statistical analysis. there was no significant difference in the positive rate of poliomyelitis antibody and gmt among the groups before immunization. there was no significant difference in antibody seroconversion rate and gmt between the same immunization procedure in dc group and liquid group after immunization. there was no significant difference in the positive conversion rate of type i and type iii antibody between 12 groups. the gmt of type iii antibody was higher than that of other groups after immunization with 2ipv+bopv, and the seroconversion rate of type ii antibody and gmt were significantly increased after immunization with 2 doses of ipv. there was no significant difference in seroconversion rate of poliomyelitis antibodies between cipv/opv sequential immunization group and sipv/opv sequential immunization group, but the serum antibodies gmt of type i in sipv/opv sequential immunization group was higher than that in cipv/opv sequential immunization group, but the serum antibodies gmt of type ii in was lower than that in cipv/opv sequential immunization group. after 2 doses of ipv, the gmt of type ii antibody in sipv/opv group was significantly increased , and there was no significant difference comparison with cipv/opv group. there was no significant difference in type iii antibody gmt between cipv/opv group and sipv/opv group. in each immunization program group, the detoxification rate reached a peak within 7 days after opv vaccination and then decreased gradually. the detoxification rate of any type of virus appeared within 3 weeks after the first dose of opv. the detoxification rate of type iii poliovirus was the highest. and there was no significant difference in the detoxification rate of type i and type ii poliovirus. the detoxification rate of type iii poliovirus in sipv+2bopv group was higher than 2sipv+bopv group and 2sipv+topv group; cipv+2bopv liquid group was higher than 2cipv+topv group.  the positive samples detected by cell culture method can be detected by real-time quantitative pcr, and more positive results can be detected by real-time quantitative pcr. there was no significant difference in the typing of poliovirus in cell culture by neutralization test, elisa and rt-pcr.

conclusion the basic immunization effect of polio vaccine was affected by different sequential immunization procedures, and the immune effect of two doses of ipv was better than that of one dose of ipv, especially type ii. the sequential immune detoxification of different poliomyelitis was different. the increase of ipv dosage could not significantly reduce the detoxification rate, but could reduce the detoxification time of each type of poliovirus.the detection rate of poliovirus in original fecal samples by real-time quantitative pcr was higher than that by cell culture, but it was unable to judge whether the virus have infectious or not. therefore, when the number of the sample is large, it can be used to screen out the positive samples by real-time quantitative pcr, and then use the cell culture method to detect.the results of neutralization test, elisa and rt-pcr are very consistent. the latter two methods can be used instead of the classical neutralization test to identify the type of poliovirus in cell culture.

key words: sabin inactivated poliomyelitis vaccine (sipv); oral poliovirus vaccine(opv); sequential immunization; detoxification