Characterization of mouse-hamster somatic cell hybrids by PCR: A panel of mouse-specific primers for each chromosome

Mouse/hamster somatic cell hybrids form a valuable resource for mouse gene mapping. Characterization of these hybrids by isozyme analysis can be technically demanding and time-consuming. Species-specific polymerase chain reaction (PCR), where a mouse gene but not its homolog in the hamster is amplified, can provide an alternative means of characterization. Mouse-specific primers have been designed for at least one gene on each of the mouse autosomes and the X Chromosome (Chr). Primers are chosen to correspond to untranslated regions of the mouse gene concerned, in order to decrease the chance of crosshybridization with the homologous hamster gene. These primer sequences are presented, together with the conditions for their use.     

大数量序列的PCR保守引物设计实践

引物设计前的序列的全面检索,未注释序列的归类,经多序列比对求带有模糊碱基代码标识(IUPAC ambiguity codes)的共有序列,对设计高质量的引物至关重要,是引物设计过程的难点。目前,综合性核酸序列分析软件,单功能应用软件,在解决上述问题时均显不足。应用互联网提供的在线生物应用程序实践了一种多程序组合使用设计大数量序列的保守引物的方法,探讨了实现大数量序列的保守引物设计的一般流程。     

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.     

PCR detection of DNAs of animal origin in feed by primers based on sequences of short and long interspersed repetitive elements

PCR primers for the detection of materials derived from ruminants, pigs, and chickens were newly designed on the basis of sequences of the Art2 short interspersed repetitive element (SINE), PRE-1 SINE, and CR1 long interspersed repetitive element (LINE), respectively. These primers amplified the SINE or LINE from total DNA extracted from the target animals and from test feed containing commercial meat and bone meal (MBM). With the primers, detection of Art2, PRE-1, or CR1 in test feed at concentrations of 0.01% MBM or less was possible. This method was suitable for the detection of microcontamination of feed by animal materials.     

Analysis of common wheat varieties and near-isogenic lines by PCR with allele-specific primers for Gli-1 and Glu-3 loci

The allelic characteristics of the Gli-A1, Gli-B1, Gli-D1 and Glu-A3 loci of 14 bread wheat varieties and 6 near-isogenic lines derived from Bezostaya 1 have been detected by PCR analysis. The conformity of molecular-genetic data and electrophoresis of storage proteins has been determined: the allelic variants of gliadins Gli-A1o and Gi-A1m correspond to the PCR-allele GliA1.2, the gliadin variants Gli-A1f, Gli-A1b, Gli-A1c correspond to the PCR-allele GliA 1.1, the allelic variants Gli-B1b, Gli-B1d--to the PCR-allele GliB1.1 and the variants Gli-B1e, Gli-B1g, Gli-B1c-to the PCR-allele GliB1.2. A new PCR-allele at the GliB) locus in the line Gli-B1-12 (with the gliadin block Gli-B1o from Levent) was identified.     

Genus- and species-specific PCR primers for the detection and identification of bifidobacteria

16SrDNA-targeted genus- and species-specific PCR primers have been developed and used for the identification and detection of bifidobacteria. These primers cover all of the described species that inhabit the human gut, or occur in dairy products. Identification of cultured bifidobacteria using PCR primer pairs is rapid and accurate, being based on nucleic acid sequences. Detection of bifidobacteria can be achieved using DNA extracted from human faeces as template in PCR reactions. We have found that, in adult faeces, the Bifidobacterium catenulatum group was the most commonly detected species, followed by Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum. In breastfed infants, Bifidobacterium breve was the most frequently detected species, followed by Bifidobacterium infantis, B. longum and B. bifidum. It was notable that the B. catenulatum group was detected with the highest frequency in adults, although it has often been reported that B. adolescentis is the most common species. Real-time, quantitative PCR using primers targeting 16S rDNA shows promise in the enumeration of bifidobacteria in faecal samples. The approach to detect the target bacteria with quantitative PCR described in this review will contribute to future studies of the composition and dynamics of the intestinal microflora.     

Improved PCR primers for the detection and identification of arbuscular mycorrhizal fungi

A set of PCR primers that should amplify all subgroups of arbuscular mycorrhizal fungi (AMF, Glomeromycota), but exclude sequences from other organisms, was designed to facilitate rapid detection and identification directly from field-grown plant roots. The small subunit rRNA gene was targeted for the new primers (AML1 and AML2) because phylogenetic relationships among the Glomeromycota are well understood for this gene. Sequence comparisons indicate that the new primers should amplify all published AMF sequences except those from Archaeospora trappei. The specificity of the new primers was tested using 23 different AMF spore morphotypes from trap cultures and Miscanthus sinensis, Glycine max and Panax ginseng roots sampled from the field. Non-AMF DNA of 14 plants, 14 Basidiomycota and 18 Ascomycota was also tested as negative controls. Sequences amplified from roots using the new primers were compared with those obtained using the established NS31 and AM1 primer combination. The new primers have much better specificity and coverage of all known AMF groups.     

Simultaneous detection of seven mutations with seven forward primers and one common reverse primer in a single PCR step

A new PCR method is described here to amplify simultaneously several fragments sharing the same template in a single PCR step with a hemi-primer. Using this method, we successfully detected various point mutations in tissue plasminogen activator (tPA) mutants.     

Absolute quantitation of specific mRNAs in cell and tissue samples by comparative PCR.

A comparative PCR assay, for the absolute quantitation of specific mRNAs in cell and tissue samples, has been designed to overcome problems with previous techniques. cDNAs made from the RNAs are co-amplified with "competitor" plasmid templates under conditions in which reagents are not limiting at the equivalence point, thereby preventing competition between target and competitor templates and distinguishing the assay from competitive PCR assays. The cDNAs are serially diluted, and competitor templates concentrations are kept constant, rather than vice versa, as occurs in competitive PCR assays. Products from target and competitor templates are resolved by electrophoresis and measured by phosphorescent or fluorescent imagery. Both products are measured to minimize errors in the competitor:target ratio. A synthetic external standard RNA is included in the tissue lysis solution and co-purified with endogenous mRNAs, thereby being subjected to identical losses of yield during subsequent procedures. The determination of the number of copies of external standard cDNA allows inefficiencies of RNA extraction and cDNA synthesis to be taken into account. Standard concentrations of plasmids containing the endogenous target sequences are also measured, so that corrections can be made for discrepancies due to unequal amplification of target and competitor sequences. These corrections, together with the use of an external standard and the PCR conditions chosen, allow for the accurate, specific and sensitive determination of the absolute number of mRNA copies in a sample.     

Development of species-specific primers for detection of Streptococcus mutans in mixed bacterial samples

Streptococcus mutans is the major microbial pathogen associated with dental caries in children. The objectives of this study were to design and evaluate species-specific primers for the identification of S. mutans. Validation of the best primer set, Sm479F/R, was performed using seven S. mutans reference strains, 48 ATCC non-S. mutans strains, 92 S. mutans clinical isolates, DNA samples of S. mutans-Streptococcus sobrinus or S. mutans-Streptococcus sanguinis, and mixed bacterial DNA of saliva samples from 33 18-month-old children. All of the S. mutans samples tested positive, and no PCR products were amplified from members of the other streptococci or nonstreptococci strains examined. The lowest detection level for PCR was 10(-2) ng of S. mutans DNA (c. 4.6 x 10(3) copies) in the test samples. The results of this study suggest that the Sm479F/R primer pair is highly specific and sensitive for identification of S. mutans in either purified or mixed DNA samples.     

Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: I—Comparative fundamental cryobiology of multiple mouse embryonic stem cell lines and the implications for embryonic stem cell cryopreservation protocols

The post-thaw recovery of mouse embryonic stem cells (mESCs) is often assumed to be adequate with current methods. However as this publication will show, this recovery of viable cells actually varies significantly by genetic background. Therefore there is a need to improve the efficiency and reduce the variability of current mESC cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of four mESC lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1 mESCs) through a comparative study characterizing the membrane permeability characteristics and membrane integrity osmotic tolerance limits of each cell line. In the companion paper, these values were used to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures, and then these predicted optimal protocols were validated against standard freezing protocols.     

食品中克罗诺杆菌属双重PCR检测试剂盒的评价

【背景】在食品安全领域,克罗诺杆菌属于需要重点监测的致病菌,当前随着分子检测相关技术的不断发展,研制有关食品中克罗诺杆菌简便、高效的检测产品至关重要。【目的】研制克罗诺杆菌检测的双重PCR检测试剂盒并评价其用于食品中克罗诺杆菌检测的实效性。【方法】优化双重PCR反应体系,反应试剂采用冻干工艺,确立了试剂盒组成,并评价其特异性、灵敏度、重复性、保质期等性能指标。【结果】克罗诺杆菌标准菌株和分离菌株均在目标位置出现两条明显条带,非克罗诺杆菌标准菌株和分离菌株均检测为阴性,纯基因组DNA检测灵敏度为2.3×10-1 ng/μL,纯培养菌检验限为3.2×104CFU/mL;对65份食品样品进行克罗诺杆菌检测,该试剂盒检测结果与标准方法检测结果一致性较高;批内、批间检测重复率均为100%,可在42°C环境放置120 h且其检测效力不受影响,4°C保质期可长达12个月。【结论】该试剂盒性能好,检测结果稳定、可靠,适用于食品中克罗诺杆菌的快速检测。