DNA荧光染色法和培养法在支原体检测中的比较

[目的]为检测支原体寻求一种有效、快速的方法。[方法]分别运用DNA荧光染色法和培养法对原材料、细胞和生物制品中的支原体污染进行检测,并对检测结果进行比较。[结果]DNA荧光染色法能够快速、准确地检测出细胞中支原体的污染;培养法在支原体检测中需要的时间较长,但能有效地对原材料、细胞和生物制品中支原体污染进行检测。[结论]有效地检测支原体应根据待检样品的特性来筛选最合适的方法。

PCR、DNA荧光染色法与培养法在支原体检测中的比较

用PCR、DNA荧光染色法及培养法，对46株细胞培养物中污染的支原体进行检测，对实验过程及所获得的实验结果进行分析，从实验的可行性、敏感性、可靠性三方面对3种方法进行了评估分析。以便选择支原体检测方法提供参考。

DNA荧光染色和PCR技术在PRRSV冻干疫苗中支原体检测的应用

为完善猪繁殖与呼吸综合征病毒(PRRSV)冻干疫苗生产各阶段的支原体检测体系,本研究分别应用DNA荧光染色法、PCR法和病毒分离培养法检测疫苗生产中的细胞、种毒、半成品、成品,分析方法的可行性和优势。结果显示,DNA荧光染色法检测PRRSV半成品抗原液的支原体污染能够于6 d内得到结果,与其余半成品检验项目用时相近,适宜在生产中应用。而采用DNA荧光染色、PCR和病毒分离培养法对我们生产的10批PRRSV冻干疫苗成品的支原体检测结果显示,DNA荧光染色法与病毒分离培养法检测的结果一致,1批疫苗的PCR检测结果与培养法检测结果不符,即PCR法检测阳性,但DNA荧光染色和病毒分离培养法检测为阴性。由此证明,DNA荧光染色法和PCR法检测疫苗中支原体的结果可靠,而PCR法检出率更高。DNA荧光染色法和PCR法操作简便、快速、经济,可以作为疫苗生产质量的内控标准,提高支原体的检出率,保证疫苗质量。

Fluorescent vital staining of plant sexual cell nuclei with DNA-specific fluorochromes and its application in gametoplast fusion

DNA-binding fluorochromes are often used for vital staining of plant cell nuclei. However, it is not always sure whether the cells after staining still remain in living state. We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei. These were: the cytoplasmic streaming in pollen tubes whose nuclei were stained, the simultaneous visualization of fluo-rochromatic reaction and nucleus staining in isolated generative cells, and the capability of isolated, prestained generative or sperm cells to fuse with other protoplasts. The results confirmed that 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33258 and mithramycin could be used as real vital stains, though their efficiency varied from case to case; among them DAPI showed best effect. The fluorescent vital staining technique offered a useful means fori-dentification and selection of heterokaryons in gametoplast manipulation studies.

体外细胞培养支原体的检测与清除

目的检测及清除细胞培养中支原体的污染。方法应用DNA荧光法及两步PCR法检测支原体,采用BM-Cycline与裸鼠皮下过继联合法去除支原体;结果DNA荧光法、两步PCR法支原体检测率分别为25.0%,40.0%;BM-cycline与裸鼠过继法对支原体的清除率为100.0%;结论上述方法能有效检测和清除细胞培养中支原体的污染。

动物细胞培养物中支原体污染的检测

[目的]建立检测细胞培养物中支原体快速有效的方法。[方法]从6种常见污染细胞的支原体16sRNA中选择2段高度保守的核酸序列作为引物,用PCR和DNA荧光染色法来检测实验室中25种动物细胞株。[结果]用DNA荧光染色法检测实验室中25种动物细胞株阳性率为24%,可疑阳性为16%;而用PCR法检测阳性率为36%。[结论]PCR法较DNA荧光染色法灵敏度更高、特异性更强,将2种方法结合应用效果更好。

生物制品中支原体污染的快速检测

用两步ＰＣＲ法（ＮｅｓｔｅｄＰＣＲ）及ＤＮＡ荧光染色技术（ＨｏｅｃｈｓｔＳｔａｉｎｉｎｇ），检测了８１份不同来源和批号的疫苗及一些实验用细胞株中的支原体，并对检测结果做了比较。ＰＣＲ阳性检出率为３８．３％，Ｈｏｅｃｈｓｔ阳性检出率为１９％。这些结果表明ＰＣＲ法较Ｈｏｅｃｈｓｔ染色法更敏感。

HEK-293细胞培养过程中支原体污染处理初探

为探索出HEK-293细胞培养中支原体污染的快速清除方法,通过大环内酯类和四环素类2种抗生素药物的单独、交替及联合使用对污染的HEK-293细胞进行处理,应用DNA荧光染色及PCR检测,比较处理前与处理后支原体污染的情况有无改善。检测结果显示,通过10 mg/mL浓度的大环内酯类和四环素类2种药物交替使用各4 d,处理2个周期后的HEK-293细胞中支原体污染已被清除。结果表明,通过10 mg/mL的大环内酯类和四环素类2种抗生素药物的交替使用,使HEK-293细胞培养过程中的支原体污染得到有效的控制,为细胞研究及疫苗工业生产奠定了基础。

Effects of hepatitis B virus infection on human sperm chromosomes

AIM: To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients.METHODS: Sperm chromosomes of 14 tested subjects (5healthy controls, 9 patients with HBV infection, including 1with acute hepatitis B, 2 with chronic active hepatitis B, 4with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes.RESULTS: The total frequency of sperm chromosome aberrations in HBV infection group (14.8%, 33/223) was significantly higher than that in the control group (4.3%,5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random.CONCLUSION: HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and/or induction chromosome aberrations, as well as the possibility of vertical transmission of HBV via the germ line to the next generation.

生物制品中支原体污染的快速检测

用两步PCR法（NestedPCR）及DNA荧光染色技术（HoechstStaining），检测了国内部分生物制品研究所生产的81份不同来源和批号的疫苗及一些实验用细胞株中的支原体，并对检测结果做了比较．PCR阳性检出率为38．3％，Hoechst阳性检出率为19％．这些结果表明PCR法较Hoechst染色法更敏感，同时亦提示必须重视检测与控制国内生物制品的支原体污染问题．

3种支原体检测方法的比较

目的比较PCR法、酶法和DNA荧光染色法3种支原体检测方法的灵敏度。方法支原体阳性的BSR细胞培养上清10倍系列稀释后（10-1~10-8）,接种至支原体阴性的Vero细胞上,盲传培养5代,每代每个稀释度的样品分别采用PCR法、酶法和DNA荧光染色法检测支原体,并以支原体阴性的Vero细胞作为阴性对照。结果 10倍系列稀释的阳性样品盲传1代,PCR法能检测到10-4,酶法和DNA荧光染色法能检测到10-3;盲传2代,3种方法均能检测到10-4;盲传3代后,3种方法均能检测到10-5,且检测的支原体滴度不随盲传代次的增加而增加。结论待检样本盲传3代后的支原体用3种方法均可检出,检测灵敏度一致。PCR法与酶法检测支原体准确、快速、简便易行,可作为DNA荧光染色法（支原体检测的金标准）的补充手段。

实验室快速检测细胞株支原体污染的方法研究

目的建立快速可靠的实验室检测细胞株培养中支原体污染的方法。方法采用DNA荧光染色试剂对本实验室11株细胞进行荧光染色观察与分析；同时从常见污染细胞的支原体16SrRNA中选择两段高度保守的核酸序列，作为支原体引物，采用聚合酶链反应（polymerase chain reaction，PCR）法对同一批细胞株进行检测，并对两种检测结果进行比较。结果采用DNA荧光染色法检出11株细胞中有2株细胞出现阳性，阳性率为18．2％；采用PCR方法检测11株细胞，有4株细胞出现阳性，阳性率为36．4％。通过两种方法比较可以发现：DNA荧光染色法检测结果阳性的细胞株用PCR方法检测结果也是阳性。结论上述两种方法均能有效地检出细胞株中的支原体污染，DNA荧光染色法虽较PCR法灵敏度低，但操作简便易观察；而PCR法成本较高，将两种方法结合应用，对DNA荧光染色法可疑阳性的标本再进行PCR检测，既提高阳性检出率，又节约了成本。