Comparison of the sensitivity of in vivo antibody production tests with in vitro PCR-based methods to detect infectious contamination of biological materials

Bacteria and viruses may be transmitted to laboratory rodents by contaminated biological materials such as transplantable tumours, cell lines, sera or other biological materials. Biological materials are currently being screened using the mouse or rat antibody production (MAP/RAP) test (serological testing). We decided to test and validate an alternative assay using polymerase chain reaction (PCR/realtime PCR) technology to detect viral contamination directly in biological material. The aim of this study therefore is the validation of our new PCR assays and the comparison of PCR and the MAP test. For 8/14 viruses, conventional PCR was more sensitive and more specific than the MAP test in detecting murine viruses. For 12/14 viruses, the realtime PCR was more sensitive than the MAP test. In 2/14 cases, all three detection methods had the same sensitivity. Furthermore, PCR screening clearly conforms to the principles of the 3Rs as a replacement technique because it eliminates the need for using animals to screen for murine viruses in biological material.     

Detection and elimination of contaminating microorganisms in transplantable tumors and cell lines.

As a quarantine of biological materials, we tested 96 transplantable tumors and cell lines for contamination with microorganisms in a mouse antibody production (MAP) test, enzymatic assay and microbiological culture. Contamination with lactic dehydrogenase elevating virus (LDV), mycoplasmas and Pasteurella pneumotropica was detected. A considerable difference in the contamination rate was observed between in vivo- and in vitro- propagated tumors. LDV in the tumors could be eliminated by both in vitro subculture and subpassage in nude rats. Mycoplasmas were eliminated by means of the mycoplasma-removal agent and P. pneumotropica by subpassage in mice. These results suggest that there is still a high risk of contamination in transplantable tumors and emphasizes the importance of adequate microbiological quality control.     

Investigation of xenotropic murine leukemia virus-related virus (XMRV) in human and other cell lines

Xenotropic murine leukemia virus-related virus (XMRV) was discovered in human prostate tumors and later in some chronic fatigue syndrome (CFS) patients. However, subsequent studies have identified various sources of potential contamination with XMRV and other murine leukemia virus (MLV)-related sequences in test samples. Biological and nucleotide sequence analysis indicates that XMRV is distinct from known xenotropic MLVs and has a broad host range and cell tropism including human cells. Therefore, it is prudent to minimize the risk of human exposure to infection by evaluating XMRV contamination in cell lines handled in laboratory research and particularly those used in the manufacture of biological products. Nested DNA PCR assays were optimized for investigating XMRV gag and env sequences in various cell lines, which included MRC-5, Vero, HEK-293, MDCK, HeLa, and A549, that may be used in the development of some vaccines and other cell lines broadly used in research. The sensitivity of the DNA PCR assays was <10 copies in approximately 1.8 x 10⁵ cells equivalent of human DNA. The results indicated the absence of XMRV in the cell lines tested; although in some cases DNA fragments identified as cellular sequences were seen following the first round of PCR amplification with the env primer pair.     

Testing the purity of cell cultures using clinical diagnostic PCR kits

Cell cultures of higher organisms, especially human cells, are increasingly used in medical, pharmaceutical, and scientific research. The main difficulty for cell cultures is hidden nonlethal contamination by mycoplasmas, viruses, and cross contamination with other cells. We suggest using PCR kits designed and officially employed in clinical diagnostics as an accessible and reliable method for monitoring the purity of cell cultures. We tested 50 human cell lines using commercial diagnostic systems for detection of papillomaviruses, herpes viruses, adenoviruses, Mycoplasma hominis, and total bacterial mass. No contamination was revealed in the cell lines tested. For the cell lines that contained integrated parts of viral genomes, the presence of the corresponding DNA sequences was confirmed. These diagnostic systems can be effectively used to test the purity of cell lines and for qualitative detection of possible contamination, as well as for quantitative evaluations using calculation of viral load, just as is practiced in clinical diagnostics.     

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert^® assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert^® mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert^®, indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.     

A panel of optimized primers and positive-control DNAs for PCR detection of common biological contaminants in mouse cell lines and tissue samples

PCR-based testing for infectious agents in mouse cell lines and tissues has recently been developed as an alternative to the traditional MAP test. One drawback to currently available PCR-based assays is the lack of appropriate positive controls for PCR detection of the infectious agents. When negative samples are the norm and positive controls are absent, it is very difficult to feel confident detecting infectious agents. To alleviate this problem, the authors developed a panel of primers and positive-control DNA plasmids that enable rapid testing of biological samples, such as cell lines, tissues, or animal sera, for presence of the infectious agents most damaging to mouse colonies.     

细胞培养中支原体污染的PCR检测

根据支原体16s rDNA序列,选择RemyTeyssou设计的三条寡核苷酸链,组成两套引物:P_(1-2a)能检测出细胞培养中常见的各种支原体,P_(1-2b)能检出无胆甾原体。反应可检出体系中10CFV的菌体。此法先用于对实验室人为污染支原体Vero细胞的检测,后与DNA 染色法和培养法比较,检测了49份生物样品,其中24份传代细胞,PCR检测的阳性率为58％,DNA染色法为42％,培养法为33％;三者的灵敏性比较,PCR可检出10~(-3)稀释度的阳性样品,高于其他两种方法。此PCR方法快速、灵敏、特异,适用于细胞培养中支原体污染的检测。     

Virus PCR assay panels: an alternative to the mouse antibody production test

Antibody production tests have traditionally been used to test biological materials for viral contamination. Now molecular biology techniques have emerged as an alternative. The authors compare MAP testing with PCR-based detection methods, focusing on differences in animal use, laboratory requirements, sample size, and limits of detection.     

Detection and monitoring of virus infections by real-time PCR

The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility, combined with an extremely broad dynamic range. A number of qualitative and quantitative PCR virus assays have been described, but commercial PCR kits are available for quantitative analysis of a limited number of clinically important viruses only. In addition to permitting the assessment of viral load at a given time point, quantitative PCR tests offer the possibility of determining the dynamics of virus proliferation, monitoring of the response to treatment and, in viruses displaying persistence in defined cell types, distinction between latent and active infection. Moreover, from a technical point of view, the employment of sequential quantitative PCR assays in virus monitoring helps identifying false positive results caused by inadvertent contamination of samples with traces of viral nucleic acids or PCR products. In this review, we provide a survey of the current state-of-the-art in the application of the real-time PCR technology to virus analysis. Advantages and limitations of the RQ-PCR methodology, and quality control issues related to standardization and validation of diagnostic assays are discussed.     

The depuration dynamics of oysters (Crassostrea gigas) artificially contaminated with hepatitis A virus and human adenovirus

Within the country of Brazil, Santa Catarina is a major shellfish producer. Detection of viral contamination is an important step to ensure production quality and consumer safety during this process. In this study, we used a depuration system and ultraviolet (UV) disinfection to eliminate viral pathogens from artificially infected oysters and analysed the results. Specifically, the oysters were contaminated with hepatitis A virus (HAV) or human adenovirus type 5 (HAdV5). After viral infection, the oysters were placed into a depuration tank and harvested after 48, 72 and 96 h. After sampling, various oyster tissues were dissected and homogenised and the viruses were eluted with alkaline conditions and precipitated with polyethylene glycol. The oyster samples were evaluated by cell culture methods, as well as polymerase chain reaction (PCR) and quantitative-PCR. Moreover, at the end of the depuration period, the disinfected seawater was collected and analysed by PCR. The molecular assays showed that the HAdV5 genome was present in all of the depuration time samples, while the HAV genome was undetectable after 72 h of depuration. However, viral viability tests (integrated cell culture-PCR and immunofluorescence assay) indicated that both viruses were inactivated with 96 h of seawater recirculation. In conclusion, after 96 h of UV treatment, the depuration system studied in this work purified oysters that were artificially contaminated with HAdV5 and HAV.     

Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines

Mycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15-35% of all cell lines are infected with a limited number of mycoplasma species of predominantly human, swine, or bovine origin. We examined the mycoplasma contamination status in 495 cell cultures by polymerase chain reaction (PCR) assay, microbiological culture method, and deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR and DNA-RNA hybridization, in order to determine the sensitivity and specificity of the PCR assay in routine cell culture. For those two cohorts, results for the three or two assays were concordant in 92 and 91% of the cases, respectively. The sensitivity (detection of true positives) of this PCR detection assay was 86%, and the specificity (detection of true negatives) was 93%, with positive and negative predictive values (probability of correct results) of 73 and 97%, respectively. PCR defined the mycoplasma status with 92% accuracy (detection of true positives and true negatives). The mycoplasma contaminants were speciated by analyzing the PCR amplification fragment using several restriction enzymes. Most of the cultures (47%) were infected with Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M. arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick mycoplasma detection assay that is suitable for the routine screening of cell cultures.     

Efficient DNA fingerprinting method for the identification of cross-culture contamination of cell lines

In order to identify cross-culture contamination of cell lines, we applied DNA fingerprinting using variable number of tandem repeat (VNTR) loci and short tandem repeat (STR) loci amplified by polymerase chain reaction (PCR) instead of a radioisotope labeled multilocus probe. Eleven cell lines were used for the Apo B and D1S80 loci detection, and twelve cell lines were examined in the Y-chromosome analysis. The data obtained from the sister cell lines NALM-6 and B85, two MOLM-1 cultures from two cryopreserved tubes, and four subclones of BALM-9 and its sister cell line BALM-10, displayed clear and distinct bands of each PCR product for both Apo B and D1S80. Detection of a Y-chromosome DNA sequence is another very informative marker for the identification of cell lines, if the Y-chromosome is present. We examined eight cell lines for the expression of four STR loci; the data thus generated were compared with the results previously reported from other laboratories. The resulting electrophoretic banding patterns showed that our "home-made" STR detection system is a useful and efficient tool for the authentication of cell lines. PCR detection of VNTR and STR loci represents a simple, rapid and powerful DNA fingerprinting technique to authenticate human cell lines and to detect cross-culture contamination. This PCR technique may be used in lieu of the more time-consuming, labor-intensive and radioactive Southern blot multilocus method.     

Tandem use of immunofluorescent and DNA staining assays to validate nested PCR detection of mycoplasma

Mycoplasma contamination in cell culture is a serious setback to cell culturists across the world with a very high rate of reported occurrence particularly because of difficult early detection. Out of a variety of detection methods known, the double-step nested polymerase chain reaction (PCR)-based detection of mycoplasma in cell culture has been critically viewed upon because of chances of producing reliable results. A nested PCR technique, described to detect a large range of cell-culture-contaminating mycoplasma species, with greater sensitivity to detect as low a contamination as a few organisms, was compared with the results from two cytological techniques employed in tandem. These are DNA staining using Hoechst, the gold standard, and an immunofluorescent assay using a highly specific monoclonal antibody. The study undertaken on randomly collected cell cultures revealed a false-negative and several false-positive results in comparison to the cytological methods employed. The observations were particularly more unambiguous with the immunofluorescent assay employed in the study while simultaneously employed Hoechst staining serving as an indicator of bacterial contamination. There is a general apprehension that genus-specific PCR approaches could be associated with inaccurate outcome and only species-specific PCRs may be satisfactory in routine screening for mycoplasma contamination in cell cultures. At this juncture, it may be suggested that caution must be exercised while adopting the two-step nested PCR-based detection approaches, and the simultaneous employment of cytological methods used in this investigation could prove to be practicable in the proper interpretation of results.     

Microtubule-associated Proteins 1 (MAP1) Promote Human Immunodeficiency Virus Type I (HIV-1) Intracytoplasmic Routing to the Nucleus

After cell entry, HIV undergoes rapid transport toward the nucleus using microtubules and microfilaments. Neither the cellular cytoplasmic components nor the viral proteins that interact to mediate transport have yet been identified. Using a yeast two-hybrid screen, we identified four cytoskeletal components as putative interaction partners for HIV-1 p24 capsid protein: MAP1A, MAP1S, CKAP1, and WIRE. Depletion of MAP1A/MAP1S in indicator cell lines and primary human macrophages led to a profound reduction in HIV-1 infectivity as a result of impaired retrograde trafficking, demonstrated by a characteristic accumulation of capsids away from the nuclear membrane, and an overall defect in nuclear import. MAP1A/MAP1S did not impact microtubule network integrity or cell morphology but contributed to microtubule stabilization, which was shown previously to facilitate infection. In addition, we found that MAP1 proteins interact with HIV-1 cores both in vitro and in infected cells and that interaction involves MAP1 light chain LC2. Depletion of MAP1 proteins reduced the association of HIV-1 capsids with both dynamic and stable microtubules, suggesting that MAP1 proteins help tether incoming viral capsids to the microtubular network, thus promoting cytoplasmic trafficking. This work shows for the first time that following entry into target cells, HIV-1 interacts with the cytoskeleton via its p24 capsid protein. Moreover, our results support a role for MAP1 proteins in promoting efficient retrograde trafficking of HIV-1 by stimulating the formation of stable microtubules and mediating the association of HIV-1 cores with microtubules.     

Cell lines authentication and <Emphasis Type="Italic">mycoplasma</Emphasis> detection as minimun quality control of cell lines in biobanking

Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.     

A robust method for bacterial lysis and DNA purification to be used with real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk

A possible mode of transmission for the ruminant pathogen Mycobacterium avium subsp. paratuberculosis (MAP) from cattle to humans is via milk and dairy products. Although controversially, MAP has been suggested as the causative agent of Crohn's disease and its presence in consumers' milk might be of concern. A method to detect MAP in milk with real-time PCR was developed for screening of bulk tank milk. Pellet and cream fractions of milk were pooled and subjected to enzymatic digestion and mechanical disruption and the DNA was extracted by automated magnetic bead separation. The analytical sensitivity was assessed to 100 organisms per ml milk (corresponding to 1-10 CFU per ml) for samples of 10 ml. The method was applied in a study of 56 dairy herds to compare PCR of farm bulk tank milk to culture of environmental faecal samples for detection of MAP in the herds. In this study, 68% of the herds were positive by environmental culture, while 30% were positive by milk PCR. Results indicate that although MAP may be shed into milk or transferred to milk by faecal contamination, it will probably occur in low numbers in the bulk tank milk due to dilution as well as general milking hygiene measures. The concentration of MAP can therefore be assumed to often fall below the detection limit. Thus, PCR detection of MAP in milk would be more useful for control of MAP presence in milk, in order to avoid transfer to humans, than for herd prevalence testing. It could also be of value in assessing human exposure to MAP via milk consumption. Quantification results also suggest that the level of MAP in the bulk tank milk of the studied Danish dairy herds was low, despite environmental isolation of MAP from the herds.     

Profiling and authentication of human cell lines using short tandem repeat (STR) loci: Report from the National Cell Bank of Iran.

Assurance of cell line homogeneity and capability of cell contamination detection are among the most essential steps of cell based research. Due to high discriminatory efficiency, low cost and reliability, analysis of short tandem repeats (STR) has been introduced as a method of choice for human cell line authentication. In the present study 13 Combined DNA Index System (CODIS) based STRs along with the gender determination (Amelogenin) gene were utilized to establish a reproducible approach for the authentication of 100 human cell lines deposited in the National Cell Bank of Iran (NCBI), using the polymerase chain reaction (PCR) method. PCR products were subsequently analyzed by polyacrylamide gel electrophoresis (PAGE) and visualized by silver staining followed by gel documentation and software analysis. STR profiles obtained were compared with those of the American Type Culture Collection (ATCC) and the Japanese Collection of Research Bioresource (JCRB) as STR references. We detected 18.8% cross contamination among the NCBI human cell lines. To our knowledge, this is the first report of authentication of human cell lines using the 13 CODIS core STRs combined with Amelogenin.     

Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination

In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.