细胞培养中细菌类微生物污染的快速检测

目的 检测细胞培养中细菌类(包括支原体)微生物的污染情况。方法 用细胞培养中常见的培养物人为污染He-La细胞，设计细菌类微生物16S rRNA基因序列通用引物，PCR扩增16SrRNA基因序列片断，建立PCR检测培养细胞污染的方法，并用该方法检测本室保存的细胞株的污染情况。结果 人为污染绿脓杆菌、大肠杆菌、白色葡萄球菌和支原体M.fernentans的Hela细胞的培养上清中均扩增出大小的片段，与目的片段相符。本室所收藏的15个细胞株有3株的培养上清中扩增出大小的片段。结论 本实验建立了：16SrRNA基因序列通用引物PCR法，可用于快速检测培养细胞中细菌类微生物的污染。     

两步PCR法检测细胞培养物中支原体污染的初步结果

本文报道用两步ＰＣＲ法快速检测细胞培养物中污染的支原体（Ｍｙｃｏｐｌａｓｍａ）。两套引物选自１６ＳｒＲＮＡ和２３ＳｒＲＮＡ保守区域，外部引物ＰＦ１，ＰＲ１扩增的ＤＮＡ片段在３６０～５００ｂｐ之间，内部引物ＰＦ２，ＰＲ２扩增的ＤＮＡ片段在１４０～２２０ｂｐ之间。试验表明，两步ＰＣＲ法能检出污染细胞培养物七种常见的支原体。１７份待检细胞培养物，两步ＰＣＲ法检测，９份（５２．９％）为阳性，直接培养法检测，５份（２９．４％）为阳性。采用快速热循仪，使ＰＣＲ反应时间大大缩短，３０次循环仅需２０分钟。反应液的用量也仅１０μｌ，提高了检测效率，节省了材料，降低了试验用费。本文就两步ＰＣＲ的灵敏度等特性也作了初步测试。     

用一种单克隆抗体快速简易地检出细胞培养中的支原体(文摘)

检测支原体污染是细胞培养中有待解决的一个问题。作者应用常见几种污染细胞培养的支原体(口腔支原体、精氨酸支原体、猪鼻支原体,莱氏无胆甾原体及唾液支原体,几种支原体占污染细胞培养支原体的96％)。为抗原,免疫了雌性BALB/c小鼠。将小鼠脾细胞与骨髓瘤细胞Xg3Ag 865融合成杂交瘤细胞,建立了30种抗支原体单克隆抗体杂交瘤细胞等。应用ELISA,     

几种防治组织培养中支原体污染方法的探索

本文报告了对几种防治组织培养中的支原体污染的方法的研究结果。试用各种滤器过滤血清和56℃灭活方法,以去除小牛血清中污染的支原体。采用半固体培基方法鉴定支原体结果证明,0.22u微孔滤膜和EKS蔡氏滤板可以去除小牛血清中污染的支原体。但G6玻璃滤器和0.3μ微孔滤膜过滤不能清除血清中的支原体,56℃加热30分钟,重复三次不能使血清中的支原体灭活。对于已经污染有支原体的传代细胞株,使用各种抗生素或在培养物中加入小鼠腹腔饲养细胞都不能去除支原体。但是将污染细胞接种于小鼠腹腔,经过一定时间再取腹水或实体瘤细胞培养,鉴定结果表明,通过小鼠腹腔内培养的NS-1细胞,不仅清除了污染的支原体,用于融合也获得满意效果。     

用滚环扩增技术检测污染细胞的5种支原体

目的 建立滚环扩增技术检测5种常见的细胞污染支原体(猪鼻支原体、发酵支原体、口腔支原体、精氨酸支原体、莱氏无胆甾原体)的方法.方法 使用细胞污染支原体16S-23SrRNA间隔区公共引物(SPS1,SPA2)扩增间隔区靶基因.设计每一种支原体特异性锁式探针,锁式探针与扩增的靶基因杂交结合形成环化单链分子,加入特异性引物在Corbett RotorGeneTM 6000扩增仪滚动扩增环化单链分子,观察结果.结果 滚环扩增技术能敏感和特异的检测上述5种支原体标准株,并能敏感的检测10个拷贝的靶基因.在62个细胞培养物样本的检测中,37个样本检测出一种支原体,14个样本检测出两种支原体,11个样本为支原体阴性.滚环扩增方法检测结果与支原体种特异性PCR检测结果一致.结论 滚环扩增作为一种新的扩增技术能敏感和特异的检测细胞污染支原体.     

4种抗支原体单克隆抗体免疫学特性的研究

为了有效地控制细胞质量、我们从1987年开始、相继建立了抗口腔支原体(M.orale),精氨酸支原体(M.arginini),猪鼻支原体(M.hyorhinis).和莱氏无胆甾原体(A.laidlawii)的杂交瘤细胞系共20株。并用IFA法、BA法或APAAP法对单克隆抗体进行了检测和鉴定。单克隆抗体(McAb)亚类鉴定结果:8株为IgG1,2株为IgG2b,其余10株为IgM。抗体效价测定大多数在4000～8 000之间。利用支原体纯培养的菌落进行了IFA染色试验,封闭试验,生长抑制试验和免疫电镜检查,均证实了它们的特异性,并获得2株对同属支原体有抗原交叉的McAb(3D1和3A11)。利用上述McAb检测实验室常用细胞的支原体污染,较常规法和DNA荧光染色法简便,快速,特异性较高。     

培养细胞污染支原体的PCR检测方法的建立及应用

在生物制剂的生产和研发领域,细胞培养扮演着极其重要的角色：用原代细胞或传代细胞制造疫苗;杂交瘤细胞制造单克隆抗体;基因工程或细胞生物学的研究等。然而支原体污染常常成为细胞培养的不速之客,严重影响着细胞及生物制剂的质量。污染细胞最常见的支原体包括：     

清除杂交瘤细胞支原体污染几种方法的比较

实验室细胞培养中，支原体污染是一个普遍存在的问题，据文献报道污染率高达90％’‘’。由于受污染的支原体可干扰细胞的DNA，RNA和蛋白质的合成，消耗培养液中的氨基酸，从而影响细胞的正常生长，严重者可导致细胞死亡，因此，清除污染的支原体，具有重要的意义。检测支原体的方法有多种，但基于本实验室条件，我们采用了培养法和DNA荧光染色法检测杂交瘤细胞株污染的支原体。由于要彻底清除污染的支原体相当困难，实际工作中应预防为主，一旦发现有支原体污染就应废弃。而对某些建株困难的重要细胞株，则须设法清除污染的支原体。目…     

检测支原体污染的一种敏感的微量定量试验:IL-2依赖性细胞株的增殖抑制试验

检测细胞培养中支原体污染的方法很多,但多数方法复杂,价格昂贵或不可靠,不适作细胞培养室的常规筛选方法.因此,作者建立了一种简便、快速而敏感的IL-2依赖性细胞株的增殖抑制试验.其原理:IL-2依赖性小鼠细胞毒性T淋巴细胞株(CTLL)在IL-2饱和浓度下,对外源性胸腺嘧啶核苷利用率非常高,掺入的~3H Tdr高达100,000～200,000cpm,因此增殖迅速.然而在被支原体污染的上清液中,由于支原体在消耗丝氨酸时产生了特异性核苷磷酸化酶,它能把外源性胸腺嘧啶核苷裂解成胸腺嘧啶,从而使培养的细胞在合     

46株分泌单抗杂交瘤细胞株支原体污染的检测

４６株分泌单抗杂交瘤细胞株支原体污染的检测李秀华，郭玮，高光，范学祥，吕秀华，袁曾麟，尹红章，李德富（中国药品生物制品检定所，北京１０００５０）细胞培养物感染支原体是一个十分严重的问题，尤其是对有重要应用价值的杂交瘤细胞，往往造成细胞生长缓慢、胞浆颗...     

Mycoplasma infection of cell cultures: Isolation and detection

Summary Mycoplasma contamination of cell cultures was first reported in 1956 and continues today to be a major concern to the investigator. Although awareness has increased over the years, mycoplasma infection of cell cultures remains a common, troublesome problem that affects many cell functions and can markedly influence proper interpretation of test results. Up-dated, sensitive mycoplasma test procedures are described. These include isolation of mycoplasmas by agar and large volume broth media procedures, and detection in an indicator cell culture system with a DNA-binding fluorochrome stain. Although prevention is the first line of defense, occasionally the considerable effort required to eliminate the contaminant from an irreplaceable cell line can be justified.     

Management of mycoplasma contamination in in vitro culture of Plasmodium falciparum without antibiotic treatment – a preliminary report

The presence of foreign contamination, especially of mycoplasmas, is a major hindrance in long term in vitro cultivation of Plasmodium falciparum and may be a source of false-positive results. Efforts have been made to control mycoplasma contamination by trypsinization of P. falciparum culture. Samples of accidentally contaminated cultures were used for this study. The presence of Mycoplasma orale in contaminated culture was ascertained by a species-specific PCR-based mycoplasma detection kit (Takara; Cat. No.6601). Trypsinization was carried out using trypsin-EDTA and the growth profile of P. falciparum was monitored for more than three weeks post-trypsinization. The studies were carried out with four different P. falciparum strains, various serum supplements and human erythrocytes belonging to different blood groups. It was interesting to observe that, irrespective of the different strains of P. falciparum and the variety of serum supplements and erythrocytes, mycoplasma contamination can successfully be removed from P. falciparum culture by trypsinization. No antibiotic except gentamicin, which is routinely used, was added to the medium. Results of this study indicate that the frequent appearance of mycoplasma in continuous long-term cultures of P. falciparum can be managed by trypsinization.     

Comparative study of the sensitivity of Mycoplasma detection methods in cell cultures

The results of a comparative study of the sensitivity of several methods for mycoplasma detection in cell cultures are presented. The most sensitive method was found to be that of electron microscopy detecting mycoplasmal contamination in 100% preparations. Seeding of the material on mycoplasma-elective nutrient medium (0.3% PPLO agar) and the method of autoradiography detect the culture contamination in 90% of the test materials. Staining of cell cultures with orsein and Schiff reagent are less sensitive methods revealing mycoplasmal contamination in 69% and 77.7% of the cultures, respectively.     

Sequence homologies between Mycoplasma and Chlamydia spp. lead to false-positive results in chlamydial cell cultures tested for mycoplasma contamination with a commercial PCR assay

Mycoplasma contamination is a frequent problem in chlamydial cell culture. After obtaining contradictory contamination results, we compared three commercial PCR kits for mycoplasma detection. One kit signaled contamination in mycoplasma-free Chlamydia pneumoniae cultures. Sequencing of cloned PCR products revealed primer homology with the chlamydial genome as the basis of this false-positive result.     

Antibody-free target cells stimulate chemiluminescence in polymorphonuclear leukocytes: an artifact due to mycoplasma contamination

The generation of toxic oxygen species represents a significant mechanism in the killing of microorganisms and antibody-coated cells by phagocytic cells. We have investigated the possibility that antibody-free target cells stimulate reactive oxygen generation in polymorphonuclear leukocytes (PMNL) using the measurement of luminol-dependent chemiluminescence (CL). It was found that the induction of CL consistently correlated with a mycoplasma contamination of the target cells. Free mycoplasma organisms of two species found frequently as contaminants in cell culture also stimulated CL. Upon artificial infection with mycoplasma, cultured cells acquired the capacity to evoke CL generation in PMNL. Our experiments strongly suggest that the induction of reactive oxygen generation by antibody-free target cells is an artifact due to mycoplasma contamination of the target cell cultures.     

Detection of mycoplasma contamination through modulation (stimulation or inhibition) of thymidine incorporation by unstimulated mouse spleen cells.

In the present report we describe a rapid and sensitive assay for mycoplasma detection in cell cultures. The assay is based on the ability of contaminated culture supernatants to modulate [3H]TdR incorporation by unstimulated mouse splenocytes. Several mycoplasma species (Mycoplasma orale, culturable and non-culturable strains of Mycoplasma hyorhinis) inhibited [3H]TdR incorporation and permitted the detection of some contaminated cell cultures that would otherwise have escaped detection in assays measuring [3H]TdR incorporation by mitogen-stimulated splenocytes. On the other hand, several other mycoplasma species (Mycoplasma arginini, Mycoplasma hominis) strongly enhanced [3H]TdR incorporation by unstimulated splenocytes. This enhancement was optimally detectable on day 2 after initiation of the cultures. The sensitivity of the assay was determined for a mycoplasma species (culturable M. hyorhinis) that inhibited as well as for one (M. arginini) that enhanced [3H]TdR incorporation. In both cases, the sensitivity was such that 1-3 x 10(2) mycoplasma colony-forming units (CFU) could be detected.     

Validation of nested PCR and a selective biochemical method as alternatives for mycoplasma detection

Direct culture is the most common way to reliably detect mycoplasma, but it is not practical for the qualitative control of cell therapeutics because of the elaborate culture medium, the prolonged incubation time, and the large sample volumes. Here, we chose two alternative methods using commercial detection kits, the PCR mycoplasma detection kit with nested PCR and the selective biochemical method, MycoAlert^®, and validated them with the direct culture method as a reference. We tested eight mycoplasma species and five validation parameters: specificity, detection limit, robustness, repeatability, and ruggedness, based on the regulatory guidelines in the US Pharmacopoeia. All experiments were performed using fibroblasts spiked with mycoplasma. Specificity tests for both methods included all mycoplasma species, except Mycoplasma pneumonia and M. genitalium for the nested PCR and Ureaplasma urealyticum for the MycoAlert^® assay. Regarding the detection limit, the nested PCR proved to be as sensitive as the direct culture method and more sensitive than the MycoAlert^® assay. The predicted median for probit = 0.9 was 54 (44–76) CFU/ml for M. hyorhinis and 16 (13–23) CFU/ml for M. hominis by the nested PCR, but 431 (346–593) CFU/ml and 105 (87–142) CFU/ml, respectively, with MycoAlert^®. Changes in the concentration of reagents, reagent lot, or individual analysts did not influence the results of the examined methods. The results of this study support nested PCR as a valuable alternative for mycoplasma detection. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Detection of mycoplasma contamination lymphoblastoid cell lines by monoclonal antibodies

We have produced two monoclonal antibodies, SFR1-Myco 1 and SFR1-Myco 2, that detect Mycoplasma fermentans found to contaminate lymphoblastoid cell lines (LCL). The specificity of these monoclonal antibodies against the M. fermentans was determined by indirect immunofluorescence by demonstrating the reactivity of the monoclonal antibodies with known reference strains of mycoplasma grown on soft agar. The reactivity of these antibodies against LCL in a number of immunoassays correlates completely with the presence of mycoplasma in these cells as determined by a standard mycoplasma detection assay. Because of the potential for widespread contamination of B lymphoblastoid cell lines transformed with Epstein-Barr virus-containing supernatants obtained from marmoset cell lines contaminated with M. fermentans, these monoclonal antibodies have value as screening reagents for this mycoplasma species in LCL.     

Treatment and control of mycoplasma contamination in <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> culture

A comparative efficacy of four antibiotics, plasmocin (macrolid), Biomyc-1, -2, (tetracycline), and Biomyc-3, and Mycoplasma Removing Agent (quinolone derivatives) was determined for elimination of mycoplasma from Plasmodium falciparum culture. Presence of mycoplasma was detected using enzyme-PCR-based mycoplasma detection kit and survival of malaria parasite was determined in Giemsa's stained smear made from treated and untreated cultures. It was observed that a combination of Biomyc-1 and -2 killed malaria parasites within 24 h, whereas plasmocin and Biomyc-3 caused slow death of malaria parasite stretched over a period of 6 days. The only compound which did not kill malaria parasite and eradicated mycoplasma from P. falciparum culture was observed to be MRA.     

Calcofluor white detection of fungi in cytopathology

Calcofluor/Cellufluor (CFW) binds to fungal cell walls and causes them to fluoresce blue-green when illuminated with UV light. Retrospective and prospective studies were made to determine if CFW could be added to the Papanicolaou (PAP) stain procedure without altering diagnostic cytopathologic features while still allowing fungi to be identified. The retrospective study included 136 cytology specimens that were designated positive for fungus by PAP stain; these were stained with a 0.1% aqueous solution of CFW and examined by fluorescent microscopy. The overall agreement between the two methods in the detection of fungi was 90.4%. The incorporation of CFW into the PAP stain was tested at various points in the PAP stain sequence; optimum results were obtained when CFW was introduced after acid eosin. A total of 197 random, sequentially accessioned cytology specimens were stained with the PAP/CFW combination in the prospective study. The results indicate that detection of fungi by a combination of regular light and fluorescent microscopy was far more effective than was examination for the organisms by light microscopy alone.