OBJECTIVETo develop a rapid and effective method for genomic DNA extraction with magnetic bead-based semi-automatic system.

METHODSDNA was extracted from whole blood samples semi-automatically with nucleic acid automatic extraction system.The concentration and purity of samples was determined by UV-spectrophotometer. Orthogonal design was used to analyze the main effect of lysis time, blood volume, magnetic bead quantity and ethanol concentration on the DNA yield; also the 2-way interaction of these factors.

RESULTSLysis time, blood volume, magnetic bead quantity and ethanol concentration were associated with DNA yield (P<0.05), but no interaction existed. DNA yield was higher under the condition with 15 min of lysis time, 100 μl of blood volume, 80 μl of magnetic beads and 80 % of ethanol. A significant association was found between the magnetic bead quantity and DNA purity OD260/OD280 (P=0.008). Interaction of blood volume and lysis time also existed (P=0.013). DNA purity was better when the extracting condition was 40 μl of magnetic beads, 15 min of lysis time and 100 μl of blood volume. Magnetic beads and ethanol concentration were associated with DNA purity OD260/OD230 (P=0.017 and P<0.05), the result was better when magnetic beads was 40 μl and ethanol concentration was 80 %.

CONCLUSIONThe results indicate that the optimized conditions with 40 μl magnetic beads will generate higher quality of genomic DNA from the whole blood samples.

Analysis of Variance ; DNA ; blood ; isolation & purification ; Humans ; Immunomagnetic Separation ; methods

Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10⁻⁹ mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 10³/mL and 100% when the sperm cells number was equal to or more than 10⁴/mL. When the number of sperm cells was 10³/mL, 10⁴/mL and 10⁵/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.
Cell Separation ; DNA ; Humans ; Immunomagnetic Separation ; Lipocalins ; Male ; Polymerase Chain Reaction ; Spermatozoa

OBJECTIVETo isolate stem cells from human exfoliated deciduous teeth (SHEDs) and identify their phenotypes and multi-lineage differentiation potential.

METHODSHuman pulp tissue from exfoliated deciduous teeth were dissected and digested to obtain the single cell suspension. The SHEDs selected by magnetic activated cell sorting system (MACS) were identified by examination of the cell morphology and growth in vitro and detection of the expressions of the cell markers. Osteogenic and adipogenic induction was performed to test the multi-lineage differentiation potential of the cells.

RESULTSSHEDs were successfully isolated from human exfoliated deciduous teeth. SHEDs showed a lower growth rate than dental pulp cells and displayed high expressions of CD29 and CD105 but low expressions of CD34 and CD45 as shown by flow cytometry. Experiments of in vitro induction demonstrated a strong potential of the STRO-1+ SHEDs for osteogenic and adipogenic differentiation.

CONCLUSIONImmunomagnetic bead selection can be used to isolate and purify SHEDs, and the STRO-1+ SHEDs show the characteristics of stem cells with multipotent differentiation potentials.

Cell Separation ; Cells, Cultured ; Dental Pulp ; cytology ; Humans ; Immunomagnetic Separation ; methods ; Stem Cells ; cytology ; Tooth, Deciduous ; cytology

DNA extraction is a basic technology of molecular biology. The purity and the integrality of DNA structure are necessary for different experiments of gene engineering. As commonly used materials in the clinical detection, the fast, efficient isolation and extraction of genomic DNA from peripheral blood mononuclear cells is very important for the inspection and analysis of clinical blood. At present, there are many methods for extracting DNA, such as phenol-chloroform method, salting out method, centrifugal adsorption column chromatography method (artificial methods), magnetic beads (semi-automatic method) and DNA extraction kit. In this article, a brief review of the principle for existing DNA blood extraction method, the specific steps and the assessment of the specific methods briefly are summarized.
DNA ; isolation & purification ; Genomics ; Humans ; Immunomagnetic Separation ; Leukocytes, Mononuclear ; chemistry ; Molecular Biology ; methods ; Phenol

In order to develop a rapid method which can check Campylobacter jejuni in animal and poultry foods nicely, an immunomagnetic capture-fluorescent PCR (IMC-FPCR) method was established in this paper. The reported method involves isolation of the target pathogen by immunocapture prior to the fluorescent PCR step, therefore the immunomagnetic-beads for Campylobacter were developed, and two groups of primer/probe, which targeted for the species special sequence of flaA gene and hipO gene for Campylobacter jejuni were designed. The immunomagnetic capture-fluorescent PCR assay amplification of the hipO gene and flaA gene for detection of Campylobacter jejuni was firstly reported in this paper. Result indicated that IMC-FPCR method permits direct detection of the pathogen without an enrichment step and can be performed in approximately 24 h. The assay results are positive for all of the isolates of Campylobacter jejuni (3 isolates, including type strain ATCC 33560 and ATCC8341) with a detection limit of approximately 10 cfu/mL, are negative for Campylobacter coli and several other bacteria. IMC-FPCR assay provide not only a rapid, sensitive method for quantitative detection of Campylobacter jejuni, but also an important method for detecting of Campylobacter jejuni of viable but non-culturerable (VNC) state.
Campylobacter jejuni ; genetics ; isolation & purification ; Fluorescence ; Immunomagnetic Separation ; methods ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

Cryptosporidium can cause gastrointestinal diseases worldwide, consequently posing public health problems and economic burden. Effective techniques for detecting contaminated oocysts in water are important to prevent and control the contamination. Immunomagnetic separation (IMS) method has been widely employed recently due to its efficiency, but, it is costly. Sucrose floatation technique is generally used for separating organisms by using their different specific gravity. It is effective and cheap but time consuming as well as requiring highly skilled personnel. Water turbidity and parasite load in water sample are additional factors affecting to the recovery rate of those 2 methods. We compared the efficiency of IMS and sucrose floatation methods to recover the spiked Cryptosporidium oocysts in various turbidity water samples. Cryptosporidium oocysts concentration at 1, 10(1), 10(2), and 10(3) per 10 microliter were spiked into 3 sets of 10 ml-water turbidity (5, 50, and 500 NTU). The recovery rate of the 2 methods was not different. Oocyst load at the concentration < 10(2) per 10 ml yielded unreliable results. Water turbidity at 500 NTU decreased the recovery rate of both techniques. The combination of sucrose floatation and immunofluorescense assay techniques (SF-FA) showed higher recovery rate than IMS and immunofluorescense assay (IMS-FA). We used this SF-FA to detect Cryptosporidium and Giardia from the river water samples and found 9 and 19 out of 30 (30% and 63.3%) positive, respectively. Our results favored sucrose floatation technique enhanced with immunofluorescense assay for detecting contaminated protozoa in water samples in general laboratories and in the real practical setting.
Animals ; Cryptosporidium/*isolation & purification ; Fluorescent Antibody Technique/*methods ; Immunomagnetic Separation/*methods ; *Oocysts ; Parasitology/*methods ; Sensitivity and Specificity ; Water/*parasitology

Fetal nucleated red blood cells (nRBCs) are rare in maternal circulation, but their presence constitutes a potential source of non-invasive prenatal genetic diagnosis. This study was undertaken to establish a non-invasive prenatal genetic diagnosis method using isolated fetal nRBCs. A multi-step method including triple density gradient and magnetic activated cell sorting (MACS) using CD45 and CD71, cytospin centrifugation, K-B staining, and glycophorin A-immuno fluorescence in situ hybridization (GPA-immuno FISH) was performed. The study population included 65 patients from 8 to 41 weeks of gestation, and fetal nRBC was separated from all cases. The number of fetal nRBCs retrieved was 12.8 +/- 2.7 in 8 to 11 gestational weeks, 15.2 +/- 6.5 in 12 to 18 gestational weeks, 16.4 +/- 6.5 in 19 to 23 gestational weeks, 10.6 +/- 3.2 in 24 to 28 gestational weeks, and 5.5 +/- 1.9 in 35 to 41 gestational weeks: the mean number of nRBCs collected from 20 ml of maternal peripheral blood was 13.7 +/- 6.2. The highest value of yield was 45.6% from 12 to 18 weeks gestation. The fetal sex determination confirmed by amniocentesis or chorionic villus sampling showed 100% sensitivity and 91.7% specificity for males; 91.7% sensitivity and 100% specificity for females. We showed that fetal cells can be reliably enriched from maternal blood and that they can be used for detecting specific chromosomes by FISH with a specificity superior to current non-invasive methods.
Erythrocytes/immunology* ; Female ; Fetal Blood/immunology* ; Gestational Age ; Glycophorin ; Human ; Immunomagnetic Separation ; Immunophenotyping ; In Situ Hybridization, Fluorescence* ; Pregnancy ; Prenatal Diagnosis*

About 30%-50% of colorectal cancer patients would develop recurrence and metastasis. At present, there is still a lack of effective evaluation method for recurrence, metastasis and prognosis. In recent years, a great progress about circulating tumor cells (CTC) in diagnosis and treatment of colorectal cancer has been made. The most common CTC detection methods include immunocytochemistry, flow cytometry, PCR, immunomagnetic separation, optical fiber array scanning and CTC chip. Based on present studies, researchers reach the consensus that CTC is clinically valuable in the following aspects: detection of occult metastasis, monitor of disease progress and evaluation of response to treatment. With recent development of clinical specialization, multi-disciplinary treatment (MDT), gene detection and targeted therapy, individualized treatment may greatly improve overall survive and disease-free survival of colorectal cancer patients. However, the methods above depend on tumor tissues that are always impractical to obtain for late stage and non-surgery patients. Moreover, the size of specimen is always small, making gene expression and mutation detection difficult. CTC detection may solve such problems based on molecular biology with high plausibility and repeatability. Therefore, CTC detection can be used as a new diagnosis tool. It is believed that CTC detection will play an important role in early diagnosis, evaluating recurrence, metastasis, making individualized treatment and predicting prognosis.
Colorectal Neoplasms ; diagnosis ; Disease-Free Survival ; Flow Cytometry ; Humans ; Immunomagnetic Separation ; Neoplasm Recurrence, Local ; Neoplastic Cells, Circulating ; Prognosis

To evaluate the separation of T lymphocyte subsets by immunomagnetic beads and to find optimization of strategy for specific binding of antibody-coated beads to cells, two strategies to isolate enriched T lymphocyte subpopulation CD4+ T cells and CD8+ T cells from small volumes (< 5 ml) of peripheral blood by using immunomagnetic beads or complement cytotoxicity method were compared. The purity and activity of CD4+ T cells and CD8+ T cells were measured by using flow cytometry, trypan-blue dye exclusion test, etc. The results showed that the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using immunomagnetic beads were (94.2 +/- 1.4)% and (93.8 +/- 3.0)% respectively, higher than those of control group and the group of using completement cytotoxicity method (P < 0.05). At the same time, the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using complement cytotoxicity method were (76.0 +/- 2.8)% and (77.0 +/- 3.0)% respectively, higher than those of unenriched group (P < 0.05). The trypan-blue dye exclusion test confirmed that there were no influences on activity of CD4+ T cells and CD8+ T cells when immunomagnetic beads were used for separation of these cells from peripheral blood. It is concluded that the immunomagnetic bead method has a higher efficiency for separation of CD4+ T cells and CD8+ T cells from peripheral blood than complement cytotoxic method, especially for small sample. This method has no influence on activity and proliferation of T lymphocyte subpopulations, and would be expected to establish conditions for research of biological characteristics of CD4+ T cells and CD8+ T cells in future.
CD4-Positive T-Lymphocytes ; cytology ; CD8-Positive T-Lymphocytes ; cytology ; Flow Cytometry ; Humans ; Immunomagnetic Separation ; methods ; Leukocytes, Mononuclear ; cytology ; immunology

This study was to aimed investigate the influence of immunomagnetic sorting on detecting the genetic aberrations of multiple myeloma (MM) by interphase fluorescence in situ hybridization (FISH) and to explore the detection method suitable to use in our country. The genetic aberrations of immunomagnetically sorted and unsorted bone marrow cells from the same MM patients were detected by interphase FISH and the detectable rate of genetic aberration was compared. The types of probes included 13 q14 (RB-1) and 14q32 (IGH). The 42 and 22 sorted and unsorted marrow samples from MM patients were detected by using 13q14 probe and 14q32 probes respectively, the results indicated that the 13q14 deletion was found in 9 of 42 (21.4%) unsorted marrow samples and in 25 of 42 (56.8%) CD138(+)-sorted marrow samples. The 13q32 rearrangement was found in 7 of 22 (31.8%) unsorted marrow samples and in 14 of 22(63.6%) CD138(+)-sorted marrow samples. Both of the difference was statistically significant (p = 0.001 and p = 0.035 respectively). Percentages of cytogenetic alterations detected in unsorted bone marrow cells correlated positively with percentage of plasma cells tested by bone marrow smears or flow cytometry. When percentage of plasma cells tested by bone marrow smears exceed 50%, or by flow cytometry exceed 10%, there was no difference between 2 methods. It is concluded that immunomagnetic sorting of CD138(+) cells increases the probability of detection of the 13q14 deletion and 14q32 rearrangement in bone marrow samples. The low detectable rate of genetic aberration in unsorted bone marrow cells is associated to the low percentage of plasma cells in bone marrow samples, higher percentage of plasma cells can partly overcome the shortage of unsorted detection method. When percentage of plasma cells tested by bone marrow smears exceed 50%, or by flow cytometry exceed 10%, there was no difference between 2 methods.
Cytogenetic Analysis ; methods ; Female ; Humans ; Immunomagnetic Separation ; In Situ Hybridization, Fluorescence ; methods ; Male ; Middle Aged ; Multiple Myeloma ; diagnosis ; genetics