中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2015年
4期
299-304
,共6页
吴雪伶%赵龙%冯建平%樊金萍%赵翔%孟淑芳
吳雪伶%趙龍%馮建平%樊金萍%趙翔%孟淑芳
오설령%조룡%풍건평%번금평%조상%맹숙방
猪细环病毒%荧光PCR%细胞%分型
豬細環病毒%熒光PCR%細胞%分型
저세배병독%형광PCR%세포%분형
Torque teno sus virus ( TTSuV)%Fluorescent PCR%Cells%Genotyping
目的：建立生物制品中猪细环病毒（ Torque teno sus virus，TTSuV）污染的检测方法，并进行方法学分析及初步的应用。方法针对TTSuV保守序列设计引物和探针，建立荧光PCR方法，对方法的特异性、线性、精密度、最低检测限等参数进行验证，并对试验样本对检测的干扰性进行分析。利用该方法对猪全血样品、生产用细胞及轮状病毒疫苗样品进行检测，通过建立TTSuV分型检测的PCR方法对阳性样品进行分型。结果荧光PCR法的特异性较好，与不同种属的细小病毒、猴SV40病毒及猪圆环病毒无明显的交叉反应，TTSuV1和TTSuV2荧光PCR分别在109～103拷贝／μl和109～102拷贝／μl范围内线性较好，R2值达到0．993以上，TTSuV1和TTSuV2的最低检测限分别为1×103拷贝／μl和1×102拷贝／μl，试验内和试验间的Ct的精密度CV值均小于7％，试验内病毒拷贝数的CV值小于25％，试验间CV值小于45％。细胞样品成分对病毒检测无明显的干扰性。对20份猪全血样品进行检测，8份为阳性，其中1份为TTSuV1阳性，4份为TTSuV2阳性，3份为TTSuV1／2混合感染。 TTSuV1和TTSuV2型与标准株序列同源性分别为98％～99％和98％。对细胞样品和轮状病毒疫苗进行检测，结果均为阴性。结论成功建立了TTSuV荧光PCR检测法，能够用于生物制品TTSuV污染的检测，进一步提高了生物制品的安全性。
目的：建立生物製品中豬細環病毒（ Torque teno sus virus，TTSuV）汙染的檢測方法，併進行方法學分析及初步的應用。方法針對TTSuV保守序列設計引物和探針，建立熒光PCR方法，對方法的特異性、線性、精密度、最低檢測限等參數進行驗證，併對試驗樣本對檢測的榦擾性進行分析。利用該方法對豬全血樣品、生產用細胞及輪狀病毒疫苗樣品進行檢測，通過建立TTSuV分型檢測的PCR方法對暘性樣品進行分型。結果熒光PCR法的特異性較好，與不同種屬的細小病毒、猴SV40病毒及豬圓環病毒無明顯的交扠反應，TTSuV1和TTSuV2熒光PCR分彆在109～103拷貝／μl和109～102拷貝／μl範圍內線性較好，R2值達到0．993以上，TTSuV1和TTSuV2的最低檢測限分彆為1×103拷貝／μl和1×102拷貝／μl，試驗內和試驗間的Ct的精密度CV值均小于7％，試驗內病毒拷貝數的CV值小于25％，試驗間CV值小于45％。細胞樣品成分對病毒檢測無明顯的榦擾性。對20份豬全血樣品進行檢測，8份為暘性，其中1份為TTSuV1暘性，4份為TTSuV2暘性，3份為TTSuV1／2混閤感染。 TTSuV1和TTSuV2型與標準株序列同源性分彆為98％～99％和98％。對細胞樣品和輪狀病毒疫苗進行檢測，結果均為陰性。結論成功建立瞭TTSuV熒光PCR檢測法，能夠用于生物製品TTSuV汙染的檢測，進一步提高瞭生物製品的安全性。
목적：건립생물제품중저세배병독（ Torque teno sus virus，TTSuV）오염적검측방법，병진행방법학분석급초보적응용。방법침대TTSuV보수서렬설계인물화탐침，건립형광PCR방법，대방법적특이성、선성、정밀도、최저검측한등삼수진행험증，병대시험양본대검측적간우성진행분석。이용해방법대저전혈양품、생산용세포급륜상병독역묘양품진행검측，통과건립TTSuV분형검측적PCR방법대양성양품진행분형。결과형광PCR법적특이성교호，여불동충속적세소병독、후SV40병독급저원배병독무명현적교차반응，TTSuV1화TTSuV2형광PCR분별재109～103고패／μl화109～102고패／μl범위내선성교호，R2치체도0．993이상，TTSuV1화TTSuV2적최저검측한분별위1×103고패／μl화1×102고패／μl，시험내화시험간적Ct적정밀도CV치균소우7％，시험내병독고패수적CV치소우25％，시험간CV치소우45％。세포양품성분대병독검측무명현적간우성。대20빈저전혈양품진행검측，8빈위양성，기중1빈위TTSuV1양성，4빈위TTSuV2양성，3빈위TTSuV1／2혼합감염。 TTSuV1화TTSuV2형여표준주서렬동원성분별위98％～99％화98％。대세포양품화륜상병독역묘진행검측，결과균위음성。결론성공건립료TTSuV형광PCR검측법，능구용우생물제품TTSuV오염적검측，진일보제고료생물제품적안전성。
Objective To establish an assay for the detection of Torque teno sus virus ( TTSuV) strains and to analyze its preliminary application to biologics.Methods Primers and probe were designed according to the conserved sequences.A fluorescent PCR assay for the detection of TTSuV strains was estab-lished.Several parameters including the specificity, linearity, accuracy, sensitivity and anti-interference of the established assay were verified.The fluorescent PCR assay was performed to detect the samples of por-cine blood, cell substrate and rotavirus vaccine.The porcine blood samples positive for TTSuV strains were further genotyped.Results The established fluorescent PCR assay was confirmed to have high specificity as no cross-reactions with parvovirus virus of various species, SV40 and porcine circovirus strains were detec-ted.The linear range of the assay was 1×109-1×103 copies/μl for TTSuV1 genotype and 1×109-1×102 cop-ies/μl for TTSuV2 genotype with a R2 value more than 0.993.The sensitivity of the fluorescent PCR assay was 1×103 copies/μl for TTSuV1 genotype and 1×102 copies/μl for TTSuV2 genotype.The intra-and inter-CVs were both less than 7%in Ct values and less than 25% and 45% respectively in copies.No interfer-ence was found in the detection of TTSuV nucleic acids from cell samples.8 out of 20 porcine blood samples were positive for TTSuV strains, among which one sample was positive for TTSuV1 genotype, four samples were positive for TTSuV2 genotype and the rest were positive for both TTSuV1 and TTSuV2 genotypes.Com-pared with the reference strain, strains genotyped as TTSuV1 and TTSuV2 were respectively shared 98%-99%and 98%homologies in sequences.All of the cell substrate and rotavirus vaccine samples detected by the fluorescent PCR assay were negative for TTSuV strains.Conclusion The fluorescent PCR assay for the detection of TTSuV was established successfully, the application of which would further improve the safety of biologics.
