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Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 editing of immune checkpoint genes could improve the efficacy of T cell therapy, but the first necessary undertaking is to understand the safety and feasibility. Here, we report results from a first-in-human phase I clinical trial of CRISPR–Cas9 PD-1-edited T cells in patients with advanced non-small-cell lung cancer (ClinicalTrials.gov NCT02793856 ). Primary endpoints were safety and feasibility, and the secondary endpoint was efficacy. The exploratory objectives included tracking of edited T cells. All prespecified endpoints were met. PD-1-edited T cells were manufactured ex vivo by cotransfection using electroporation of Cas9 and single guide RNA plasmids. A total of 22 patients were enrolled; 17 had sufficient edited T cells for infusion, and 12 were able to receive treatment. All treatment-related adverse events were grade 1/2. Edited T cells were detectable in peripheral blood after infusion. The median progression-free survival was 7.7 weeks (95% confidence interval, 6.9 to 8.5 weeks) and median overall survival was 42.6 weeks (95% confidence interval, 10.3–74.9 weeks). The median mutation frequency of off-target events was 0.05% (range, 0–0.25%) at 18 candidate sites by next generation sequencing. We conclude that clinical application of CRISPR–Cas9 gene-edited T cells is generally safe and feasible. Future trials should use superior gene editing approaches to improve therapeutic efficacy. In a first-in-human phase I trial of patients with advanced lung cancer, infusions of autologous T cells edited to delete the PD-1 gene via CRISPR–Cas9 were well tolerated and did not lead to severe treatment-related adverse events.

To screen the genes with significant changes in DNA methylation level in active tuberculosis patients, we used the methylation chips and expanded the sample size to verify candidate genes.① This study enrolled 9 cases of active tuberculosis patients, 3 cases of latent tuberculosis patients and 3 cases of healthy controls whose age and gender were all matched. Genome DNA was extracted from peripheral blood mononuclear cell in blood samples collected from these candidates, and bisulfite conversion treatment was then conducted. After hybridization with the Illumina HD 450K Infinium Mehtylation BeadChip, the results were compared between patients group and control group, and GO and KEGG pathway analyses were performed to evaluate the function of differentially expressed genes. ② We further enrolled 60 cases of active tuberculosis patients and 60 cases of health controls (age-and gender-matched), DNA was extracted from their peripheral blood and also followed bisulfite conversion treatment. Pyrosequencing method was used to detect the methylation levels of candidate genesCompared with healthy controls, the fragments in the patients that showed low methylation change accounted for the vast majority. Most of the methylation differential fragments (DMRs) were located in the main body region, followed by the upstream region of transcription initiation site, and the lowest DMRs distribution area was 3´UTR area. GO and Pathway analysis showed that the functions of the differentially methylated regions related genes are mainly enriched in the biological processes of the regulation of leukocyte differentiation, apoptosis, cytokine regulation and inflammatory response which are closely related to tuberculosis. There were 32 CpG sites involved in the verified 7 tuberculosis related genes, and 16 CpG locus showed significant difference (In the course of MTB infection, the methylation status of genomic DNA is altered, and most of the differentially methylated regions (DMRs) are showed status of demethylation. The expressions of

Aims: To investigate the distribution of epidermal growth factor receptor (EGFR) mutations, and explore any relationships with clinical characteristics in non-small-cell lung carcinoma (NSCLC) patients. Materials and Methods: EGFR mutations were assessed by ADx-ARMS in 261 NSCLC patients from West China Hospital of Sichuan University. Relationships between EGFR mutation and clinical characteristics were analyzed by SPSS. Results: The EGFR mutation rate was 48.7% (127/261), 19-del and L858R mutations occurred predominantly, accounting for 33.1% and 40.9%, respectively, in mutated cases. Moreover, 10.2% patients were found to carry double mutations. EGFR mutations occurred more frequently in women (57.5%) than in men (41.8%) (P=0.01), and were more frequent in non-smokers (61.2%) than in former or current smokers (31.2%) (P

To determine the targeted regulating role of has-miR-577 and has-miR-583 on the expression of fibroblast growth factor 21 (The site of has-miR-577 and has-miR-583 target genesThe double enzyme electrophoresis and sequencing results showed that the gene fragment size and sequences of the wild type (psiCHECK2

To investigate the molecular features of spinal muscular atrophy (SMA) related genes in SMA patients of Han nationality of southwest of China.We collected 62 unrelated patients of SMA and 50 unrelated healthy individuals in this study.The copy numbers of survival motor neuron gene (Of 62 patients,the copy number of SMA1-4 were 30.65% (19/62),41.94%(26/62),16.13% (10/62),11.29% (7/62),respectively.The deletion of SMN1 exon 7 accounts for 98.38% (61/62).The deletion ofThe deletion of

To determine the correlation between gene polymorphisms in Wnt signal pathway and susceptibility of Chinese Tibetan people to tuberculosis.A total of 488 active tuberculosis patients and 454 healthy subjects(control) were enrolled in this case-control study.Five single nucleotide polymorphisms (SNPs) in Wnt signal pathway (rs4135385 inThe genotype distributions of all SNPs coincided with the Hardy-Weinberg equilibrium in the 2 groups.The frequencies of genotype and allele of rs7832767 in

Over the past 20 years,clinical molecular diagnostic technology has made rapid development,and became the most promising field in clinical laboratory medicine.In particular,with the development of genomics,clinical molecular diagnostic methods will reveal the nature of clinical diseases in a deeper level,thus guiding the clinical diagnosis and treatments.Many molecular diagnostic projects have been routinely applied in clinical works.This paper reviews the advances on application of clinical diagnostic techniques in infectious disease,tumor and genetic disorders,including nucleic acid amplification,biochip,next-generation sequencing,and automation molecular system,and so on.

To determine the correlations betweenMedical records of 94 AML-M2 cases with positiveNo significant differences in the clinical symptoms,predominantly anemia,fever and hemorrhage,were found between the

To determine gene variations and genotype-phenotype correlations in Duchenne/Bayesian muscular mystrophy (DMD/BMD) patients, and the association between dystrophin gene polymorphisms and clinical phenotype.Multiplex ligation-dependent probe amplification (MLPA) was adopted to detect dystrophin gene variations in 170 patients. Sanger sequencing was performed in 3 cases with decreased peaks in MLPA results.The MLPA detected 72.94% mutations in dystrophin gene, including 62.35% (106/170) deletions, 8.82% (15/170) duplications, and 1.76% (3/170) point mutations. 64 different types of mutations were found. 75.47% of deletions occurred in the range from exon 44 to 55. Most 5' breakpoints of exonic variations were located in 2 hotspots (major hotspot: intron 43-55; minor hotspot: intron 1-20), which is different from findings of other studies. Genotype-phenotype analysis showed that the severity of DMD/BMD was associated with frame shift mutation (r = 0.640, P0.001) but not with deletions or duplications.Deletions and duplications of exon compose the main type of dystrophin gene mutations. DMD/BMD is associated with frame shift mutation.

To determine the impacts of Wnt signaling pathway products-polymorphisms of rs4135385, rs11079571 and rs7832767 located in β-catenin gene (CTNNB1), Axin gene (AXIN2), and secreted frizzled-related protein gene (SFRP1) on the risk and treatment outcomes of acute leukemia.Bone marrows (volume 1-1. 5 mL) were collected from 372 untreated patients with acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), and peripheral blood samples (2. 0 mL) were obtained from 401 healthy controls for the purpose of total DNA extraction. Polymorphisms of rs4135385, rs11079571 and rs7832767 located in CTNNB1, AXIN2 and SFRP1 were genotyped with high-resolution melting method (HRM). Chi-square analyses were performed to compare the genotype and allele distributions of the three single nucleotides (SNPs) between the leukemia patients and healthy controls. Single factor variance tests were performed to compare the differences in clinical features among different genotype groups. Complete remission (CR) rates after induction chemotherapy were also compared between different genotype groups using Chi-square tests.No significant differences were found beiween the leukemia patients and healthy controls in the frequencies of alleles and genotypes of CTNNB1 rs4135385, SFRP1 rs7832767 polymorphisms. Those with A allele in AXIN2 rs11079571 polymorphism was less likely to have acute myelomonocytic/monocytic leukemia than those with G allele (P = 0. 016, OR=0. 677, 95%CI:0. 439-0. 930). Acute bead monocyte/mononuclear cell leukemia (AML-M4/5)patients with AA genotype presented higher platelet count (P = 0. 040), and higher complete remission rate after chemotherapy (P = 0. 040), compared with the patients with AG and GG genotypes.AML-M4/5 patients have less frequency of A allele in AXIN2 rs11079571 polymorphism than healthy controls. Patients carrying A allele have higher platelet counts and higher sensitivity to chemotherapy.

To assess the correlation between JAK2-V617F mutation and complete blood counts among patients with BCR/ABL-negative myeloproliferative diseases (MPD).One hundred and ninety one patients were recruited. Retrospectively, their laboratory data were analyzed for the counts of red blood cells (RBC), white blood cells (WBC) and platelets (PLT). And the incidence of JAK2-V617F mutation was determined.There was significant difference in the incidence of JAK2-V617F mutation between patients with different cell counts (P0.01). The incidence of JAK2-V617F mutation has increased with the counts of RBC and PLT, which was the highest (92.86%) among those featuring simultaneous increase in all three series.The incidence of JAK2-V617F mutation seems to be strongly associated with variation of peripheral blood cell counts among patients with BCR/ABL-negative MPD. Variation of peripheral blood cells, particularly RBC, may be correlated with the rate of JAK2-V617F mutation.

BACKGROUND: To establish a reliable correction method for automated hemoglobin (HGB) measurement by minimizing the interference from blood high triglyceride (TG). METHODS: Fifty whole blood samples and 50 plasma samples containing variable TG concentrations were used to determine the centrifugation speed and time. Complete blood cell counts (CBCs) were performed by an automated hematology analyzer for 102 blood samples, in which high‐level TG were artificially added. The same blood samples were centrifuged at low –speed to separate the plasma from blood cells. Then the plasma was analyzed by the same analyzer. By using the two CBC results, a correction formula was established to calculate the corrected HGB, mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) values. Comparisons were also made of HGB, MCH, and MCHC values before and after correction of in‐patient individuals who received intralipid and developed lipemia. RESULTS: The percentage differences between the corrected and true values of HGB, MCH and MCHC were −0.28%, 0.06%, and −0.31%, respectively. The correlation coefficients of corrected values versus true values of HGB, MCH, and MCHC were 0.989, 0.935, and 0.717, respectively. This correction method was also effective for native lipemic samples. CONCLUSION: High blood TG level can cause blood turbidity and erroneously high HGB results by hematology analyzers commonly used in clinical laboratories. Adding a simple step of low‐speed centrifugation and measurement of HGB in the plasma fraction allows a quick correction of HGB measurement in lipemic blood samples.

To develop an estimating formula for glomerular filtration Rate (GFR) based on serum cystatin C in patients with chronic kidney disease (CKD).Clinical characteristics of 242 CKD patients were collected. The patients were randomly divided into modeling group and model validation group. The rGFR obtained from 99mTc-DTPA clearance rate was used as a reference value of GFR. s-cystatin C was detected by latex enhanced immunoturbidimetric method. Preliminary linear regression analysis followed by multiple linear regression were performed to investigate the association between s-cystatin C and rGFR. The validity of the estimation formula was tested in the model validation group in comparison with Hoek formula and Orebro formula.With standardised countdown conversion, s-cystatin showed linear correlation with rGFR, with a correlation coefficient of 0.773. The multiple correlation coefficient, determination coefficient, adjusted R square and std. error of the estimation model were 0.863, 0.745, 0.742, and 0.207, respectively. The residuals P-P probability plot analysis showed that the model residuals fitted into normal distribution with homogeneity of variance. Theeformula was: eGFR = 67/s-cystatin C +3. No significant difference was found between the distribution of eGFR and rGFR. Our formula had an accuracy of 30% and 50%, which were no less than those obtained from Hoek formula and Orebro formula. The new formula also had acceptable bias and high precision. The Bland-Altman analysis and ROC curve analysis showed good applicability of the new formula.The GFR prediction formula we established has a good prediction performance as comparised with other formulae, which could be used in measuring GFR in CKD patients.

To compare the detection sensitivity of epidermal growth factor receptor (EGFR) mutations between allele specific oligonucleotide PCR (ASO-PCR) and bi-loop probe and specific primer quantitative PCR (BPSP-qPCR).A total of 96 non-small cell lung cancer specimens were selected from West China Hospital from September 2009 to December 2010. ASO-PCR was developed to detect the presence of classical EGFR mutations. A total 39 available specimens were also tested by BPSP-qPCR.EGFR mutation detection rate was 30.2% (26/96) by ASO-PCR. The mutation rate was higher in female than in male patients [45.5% (20/44) vs. 17.3% (9/52), P = 0.003], non-smokers than smokers [44.1% (26/59) vs. 8.1% (3/37), P0.001] and adenocarcinomas than other subtypes of lung cancer [37.0% (27/73) vs. 8.7% (2/23), P = 0.01]. Among mutation negative cases by ASO-PCR, BPSP-qPCR increased the rate of detection of 19-del and L858R mutation by 10.3% (4/39) in adenocarcinomas and non-smoking subset. Overall, the mutation detection rate of BPSP-qPCR was higher than that of ASO-PCR [66.7% (26/39) vs. 41.0% (16/39), P = 0.02].BPSP-qPCR has a better detection sensitivity than that of ASO-PCR.

To investigate the sensitivity of bi-loop probe and specific primer quantitative PCR (BPSP-qPCR) in the detection of epidermal growth factor receptor (EGFR) gene mutations in non-small cell lung cancer (NSCLC).BPSP-qPCR was employed to examine the presence of mutations of EFGR exon 19 through 21. Correlation of the mutations with clinicopathological characteristics and types of tumor samples were performed.In the cohort of 265 specimens, 30.2% (80/265) mutations were found to be 19-del and/or L858R. Females (39.7%, 31/78), non-smokers (41.0%, 43/105) and adenocarcinoma patients (37.8%, 51/135) had a higher mutation rate (P0.05) among 184 patients whose profiles were available. T790M combined with 19-del and/or L858R accounted for 3.3% (6/184) of the mutations. Male metastatic tumors (29.6%, 8/27), pleural fluids of females (42.9%, 9/21) and non-smokers (40.7%, 11/27) were found to have higher percentage of 19-del and/or L858R mutations, in contrast, no mutations were found in the metastatic lesions of non-adenocarcinoma patients (P0.05).BPSP-qPCR is a robust method in detection of EGFR mutations with high consistency and sensitivity. The difference of EGFR mutations in primary tumors, metastatic lesions and pleural fluids suggests that EGFR tyrosine kinase inhibitors (EGFR-TKI) treatment may have variable treatment effects depending on the tumor sites.

To explore the molecular and epidemic characteristics of rifampin (RFP) and isoniazid (INH) resistance of mycobacterium tuberculosis (MTB) in Sichuan.GenoType reg; MTBDRplus Assay GTplus was used to examine 68 clinical isolates of MTB and 105 clinical specimens for mutations in rpoB, katG and inhA genes related to RFP and INH resistance.Of the 151 valid tests obtained, 44 (29.14%) and 26 (17.22%) showed drug resistance and multidrug resistance, respectively. Resistance to RFP and INH was found in 21.85% (33/151) and 24.50% (37/151) of the samples, respectively. The most prevalent mutations were rpoB S531L, katG S315T1 and inhA C-15T. The multidrug resistance rate in the sputum specimens was significantly higher than that in the non-respiratory samples (19.35% vs 7.41%).Drug-resistant, especially multidrug-resistant tuberculosis is highly prevalent in Sichuan. The multidrug-resistant bacteria most frequently show rpoB S531L combined with katG S315T1 mutations, suggesting the necessity of developing rapid clinical identification methods for drug-resistant MTB to control the spread of the resistant strains.

The aim of this study was to perform an association study between two single nucleotide polymorphisms (SNPs) rs2910164 G>C and rs3746444 T>C in pre-miRNA (hsa-mir-146a and hsa-mir-499) and rheumatoid arthritis (RA) in the Han Chinese population. 208 Han Chinese patients with RA and 240 healthy controls were recruited in this study. The SNPs was genotyped by polymerase chain reaction-restriction fragment length polymorphism. Anti-cyclic citrullinated peptide (anti-CCP) antibody was measured by enzyme linked immunosorbent assay and rheumatoid factor (RF) was measured by rate nephelometry. The genotype frequencies between cases and controls were compared by χ(2) analysis. No significant association between the SNPs (rs2910164 and rs3746444) and RA was observed (P = 0.631 and 0.775, respectively), and the SNPs did not show any association with the RF-positive (P = 0.631 and 0.775, respectively). However, there was a significant difference on the level of anti-CCP antibody between different genotypes in rs3746444 (P = 0.007). The heterozygote CT had significantly higher level of anti-CCP antibody compared with homozygote CC and TT (P = 0.054 and 0.003, respectively). We first investigated the association between the SNPs (rs2910164 G>C and rs3746444 T>C) in the pre-miRNA (hsa-mir-146a and hsa-mir-499) and RA in a Han Chinese population. We did not find a significant association between the SNPs and the susceptibility to RA, while the SNP rs3746444 may affect anti-CCP antibody production.

The main risk factor for chronic obstructive pulmonary disease (COPD) is cigarette smoking. However, only 10% - 20% of chronic heavy smokers develop systematic COPD. We hypothesized that the inheritance of gene polymorphisms could influence the development of COPD, which was investigated by studying two single nucleotide polymorphisms (SNP) in exon 1 of the transforming growth factor-beta1 (TGF-beta1) gene.We enrolled 219 patients with COPD as the research group and 148 healthy people as the control group, all of whom were Chinese Han people. The polymorphisms of the TGF-beta1 gene, 869T/C and 915G/C, were analyzed using the method of amplification refractory mutation system-polymerase chain reaction (ARMS-PCR).The occurrence of the TGF-beta1 gene 869T/C polymorphism in patients with COPD was significantly different from the control group (P0.05), in which the relative risk of this disease increased in cases who had the C allele (OR: 1.131, 95%CI: 1.101 - 1.539). There was no increased frequency of TGF-beta1 915G/C gene in COPD patients compared with control subjects (P0.05).The polymorphism 869T/C in TGF-beta1 gene has a significant association with disease occurrence in COPD patients and the C allele might be a risk factor. The homozygous wild-type CC of 869T/C on TGFbeta1 could be a predisposing factor in COPD and those who carry the C allele might have particularly susceptibility to developing COPD.

To obtain the population genetic data of 17 Y-chromosomal short tandem repeat (Y-STR) in the Han population in Chengdu of Sichuan Province.The 17 Y-STR loci were amplified from the blood samples of 111 unrelated Chengdu Han individuals using the AmpFlSTR Yfiler system. The PCR products were genotyped with an ABI 3130 genetic analyzer.In the loci of in DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y-GATA-H4, DYS437, DYS438, and DYS448, 3 to 8 alleles were detected in the Han population in Chengdu, and 36 alleles were detected in the locus DYS385a/b, with the minimal gene diversity (GD) value of 0.3970 (DYS391) and maximal value of 0.9561 (DYS385a/b). The DNA samples of 16 women and 7 different species of animals were amplified, but no specific products were found for the 17 Y-STR loci. No mutations of the 17 Y-STR alleles were observed in 20 father-son pairs as confirmed by autosomal STR analysis.The 17 Y-STR loci are highly polymorphic and are suitable for personal identification, paternity testing, population genetics and anthropology studies.

To investigate the proteomic characteristics of systemic lupus erythematosus (SLE) in a SLE family from Sichuan, China which consisting of 7 members with 3 SLE cases, and to find the proteins correlated with the heredity of SLE.A total of 153 serum samples were collected from 7 members including 3 SLE sisters in this SLE family, 63 individual SLE patients, as well as 83 healthy controls. The diagnosis of SLE is based on the American College of Rheumatology criteria (1997). All serum samples were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with magnetic beads technology. Serum protein profiles were obtained by MALDI-TOF-MS combined with magnetic beads in order to identify predictive biomarkers of risk of suffering SLE. The resulting spectra were analyzed with Biomarker Wizard software 3.1.0.Four discriminative mass/charge (m/z) proteins serving as pathogenic biomarkers were identified on arrays for family SLE cases versus individual SLE and healthy controls. The protein level of peak intensities at m/z of 9342.23 was significantly greater in SLE family group compared with that in individual SLE patients and healthy controls (P0.05), those of individual SLE patients were significantly greater compared with healthy controls (P0.05); the proteins level of peak intensities at m/z of 4094.03, 5905.35 and 7973.53 in SLE family group were significantly lower compared with that in individual SLE patients and healthy controls (P0.05), those of individual SLE patients were significantly lower compared with healthy controls (P0.05).The proteins of m/z of 9342.23, 4094.03, 5905.35 and 7973.53 maybe play a great role in assemble pathogenesis of SLE and predict the risk of suffering SLE. The higher protein level of m/z of 9342.23 and the lower protein level of m/z of 4094.03, 5905.35 and 7973.53, the higher risk of sufferring with SLE.

To establish a diagnostic model for systemic lupus erythematosus (SLE) using proteiomic fingerprint techology.Blood samples were collected from 64 cases of SLE, 30 cases of rheumatoid arthritis (RA), 30 cases of Sjogren's syndrome (SS), 25 cases of systemic sclerosis (SSc), as well as 83 healthy controls. Proteomic spectra of these 232 serum samples were generated by proteomic fingerprint technology. Diagnostic model was established by a machine learning algorithm called decision boosting. The sensitivity and specificity of the diagnostic model was validated with a blinded testing set.Sixty differential protein peaks (P0.05) between SLE and control subjects were indicated, 28 of them were up regulated and 32 were down regulated in SLE patients. The algorithm identified a cluster pattern segregating SLE from non-SLE with sensitivity of 91% and specificity of 92%. The discriminatory diagnostic pattern correctly identified SLE. A sensitivity of 78% and specificity of 96% for the blinded test were obtained when comparing SLE vs non-SLE.This diagnostic model using proteiomic fingerprint techology appears to be a promising tools with high sensitivity and specificity in diagnosis of SLE.

To investigate the association of the Pro12Ala variant in peroxisome proliferators-activated receptor gamma (PPAR gamma) gene with rheumatoid arthritis.The genotypes of the Pro12Ala variant in the PPAR gamma gene were determined by polymerase chain reaction-restriction fragment length polymorphism in 421 unrelated subjects of the Han population in the Sichuan Province of China, including 207 subjects with rheumatoid arthritis and 214 subjects without the disease. The clinical data were also collected and analyzed.The allele frequencies in the case and control groups were 98.79%, 95.79% for allele P and 1.21%, 4.21% for allele A; the genotype frequencies were 97.58% and 91.59% for PP, 2.42% and 8.41% for PA, and 0 for AA. The A allele frequency was much lower in the RA group than that in the control group.The above data showed that the Pro12Ala variant of the PPAR gamma was associated with rheumatoid arthritis. The A allele might be a protective factor for RA. The Pro12Ala polymorphism in the PPAR gamma gene in Sichuan Han population is similar to that in other populations in China, but different from that in European and American populations.

To investigate the genetic features of the community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) prevalent in West China.Three CA-MRSA isolates obtained in Chengdu, Sichuan, underwent SCCmec (Staphylococcal Cassette Chromosome mec) multi-PCR, Staphylococcal protein A (spa) typing and multi-locus sequence typing (MLST) method, and their Panton-Valentine leucocidin (PVL) gene was also detected.All 3 CA-MRSA isolates were positive of mecA gene (147 bp), and the other PCR product of 750 bp was confirmed to be type IVa SCCmec. Spa typing showed that the MRSA strains s29635 and s35301 were typed as t437, and the strain s19165 was typed as t008. MLST showed that the MRSA strains s29635 and s35301 were typed as ST59, and the strain s19165 was typed as ST8. The strains s19165 and s35301 were all positive for PVL gene, and the strain s29635 was negative for PVL gene.CA-MRSA clones ST8-t008 and ST59-t437 have been isolated in West China.

Blowflies found on human corpses are important for the estimation of the postmortem interval and other questions of forensic relevance. Some of these species are difficult to differentiate morphologically, and therefore a molecular method was elaborated for species identification. Here, we describe a molecular method for rapid identification of these insects. Specific insect DNA fragments were amplified using the polymerase chain reaction, followed by direct DNA sequencing of the amplification products. Analysis of the cytochrome oxidase II sequences revealed abundant phylogenetically informative nucleotide substitutions that could identify blowfly species to species group. In contrast, because of the low level of sequence divergence of sister species, the data could not distinguish among taxa from the same species group, ie, the species within the Lucilia sericata and Lucilia cuprina groups. The molecular data support the existing species group separation of the taxa within the Calliphora. Because of the speed and accuracy of current nucleotide sequencing technology and the abundant apomorphic substitutions available from mtDNA sequences, this approach enables quick identification of species used for estimation of postmortem interval.

To solve the difficulties of identification of Sarcosaphagous flies such as Lucilia sericata (Meigen) and Lucilia cuprina (Wiedemann) which could not be identified by analyzing the 278bp and 635 bp regions of the gene encoding for cytochrome oxidase subunit I and II (CO I and CO II) in mtDNA.Specimens were collected from the corpses of rabbits on the grassland in Huhhot and Chengdu, the sequences of 551 bp region of 16S rDNA of their mtDNA were analyzed, the multiple-alignment program DNAMAN(version 4.0) and MEGA 2.1 sofeware were employed for sequence alignments neighbour-joining tree construction.Lucilia sericata (Meigen) and Lucilia cuprina (Wiedemann) were distinguished successfully by sequence analysis of The 551 bp region of the gene of 16S rDNA.The 551 bp region of the gene of 16S rDNA of sarcosaphagous flies can be used for identifying them on species level effectively. It is likely to be a successful compliment to identify the sarcosaphagous flies by sequence analysis of CO I and CO II in mtDNA.

This study was based on a 278 and 635 bp region of the gene for cytochrome oxidase subunit I and I (CO I and CO II ) encoding region of mtDNA; the aim was to solve the problems in identifying Sarcosaphagous flies, particularly in the flies' larvae and eggs which could not be identified only by use of their morphologyical features.Samples of sarcosaphagous flies and larvae were collected from those on the corps of rabbits on the grassland in the Huhhot district and of a pig on the sandy ground in the Dunhuang district. The mtDNA of flies and their larvae and eggs was extracted using the Chelex technique. Polymerase chain reactions were conducted on a Perkin-Elmer 9600 thermal cycler, followed by vertical non-denaturing polyacrylamide gelectrophoresis. PCR products were purified using the Nucleic Acid Purification Kit. Sequences of both strands were obtained by direct sequencing of the double-stranded PCR product using one of the PCR primers and the ABI PRISM Big Dye Terminator Cycle Sequencing Kit. Sequence reactions were electrophoresed on ABI Model 377 DNA Sequencers. A neighbour-joining tree using the Tamura and Nei model of nucleotide substitution was constructed using the MEGA2. 1 package.A 278 and 635 bp region of the gene for CO I and CO II encoding region of mtDNA of Sarcosaphagous flies and their larvae and eggs was noted to show the percentages for the sequence divergence within species (less than 1%) and the sequence divergence between species (above 3%). For species that diverged from all others by a relatively large percentage and had small within-species variation, the least percentages of sequence divergence were given which distinguished any individual within that species from any other species.A 278 and 635 bp region of the gene for CO I and CO II encoding region of mtDNA of Sarcosaphagous flies and their larvae and eggs has been effectively used for the molecular phylogeny and the identification of their species group. CO I and CO II, or CO II, is better than CO I.

We focus on developing a multiplex PCR system for Y-STR loci that can be detected by double fluorescent system and assessing their usefulness in forensic mixture samples.The primers of four Y-STR loci (DYS-GATA-A10, DYS531, DYS557 and DYS448) amplified by multiplex PCR technique were labeled with fluorescence, then the PCR products of these Y-STRs loci were detecting and typing by ABI PRISM310 Genetic Analyzer.When 120 unrelated individuals from the Han population in Chengdu were detected by the system, Y-GATA-A10, DYS531, DYS557 and DYS448 showed 5, 5, 8, 7 alleles, respectively. A total of 78 different haplotypes was identified and the genetic diversity reached 0.9881. To the three cases of mixture stains failed by using conventional autosomal STR analysis, our multiplex system drew conforming conclusion comparing to the suspect's Y-STRs genotypes.Our results show that the multiplex system of four Y-STR will be very powerful for Y-STR database establishing, the paternity testing and mixture stains identifying.

Identifying sarcosaphagous flies specimens is an important first step in a forensic-entomological analysis. It is traditionally performed using morphological features of the Sarcosaphagous Flies. However, Morphological identification may be complicated by the numerical diversity of species and physical similarity between different species, particularly in immature stages. The sequences focused on some sections of the cytochrome oxidase I and II (CO I and CO II) encoding region of mtDNA could be as the prospective basis of a diagnostic technique. By Analysis of these sequences, forensic doctors can reveal abundant phylogenetic informative nucleotide substitutions that could effectively identify Sarcosaphagous flies to species group. It was not reported in our country before and was reviewed in this article now.

To investigate the genetic polymorphisms of three short tandem repeats loci of chromosome X in Chinese Han population in Chengdu area and its use in forensic science. Three X-chromosome linked short tandem repeat loci were analyzed by PCR followed by polyacrylamide gel electrophoresis. Hardy-Weinberg equilibrium was tested and forensic interested value was calculated . The power of exclution of DXS6804, DXS9896 and GATA144D04 is 0.5990, 0.6220, 0.4280, respectively. The result showed that all the three STR loci were polymorphic among 100 unrelated females and 120 unrelated males from Chinese Han population. Chi(2) tests demonstrated that genotype frequencies in females did not depart from Hardy-Weinberg equilibrium. Three X-chromosome linked short tandem repeat loci have high polymorphism, they can be applied to forensic medicine and population genetics.

To understand the allele structure and genetic polymorphism at three STR loci on chromosome X in Chinese Han population and make an evalution of their forensic application.EDTA-blood samples were collected from the unrelated individuals in Chengdu, China. After being extracted with Chelex method, the DNA samples were amplified by PCR technique. The PCR products were analyzed by PAG electrophoresis and the approach of automated fluorescence detection. Hardy-Weinberg equilibrium of females was tested and every forensic interested value was calculated.The polymorphisms of all 3 STR loci were obtained from 100 unrelated females and 120 unrelated males from Chinese Han ethnic group. Chi-square tests on the genotype frequencies in females did not reveal deviations from Hardy-Weinberg equilibrium.The obtained data are beneficial to understanding the population genetics of the three STR loci in Chinese Han population. For forensic genetics, the obtained data can be used to calculate the probabilities dealing with the paternity test and the individual identification.

One multiplex genotyping system was developed in using silver staining with allelic ladders for three Y-chromosome STR markers (DYS434, DYS443, and DYS456), with a view towards the application of rapid and simple genotyping assay methods for DNA profiling. The distributions of haplotypes for three Y-STR loci(DYS434, DYS443, and DYS456) was investigated in a Tibetan ethnic group of Chinese population.Allele and haplotype frequencies at these Y-STRs loci(DYS434, DYS443, and DYS456) were analysed by PCR amplification using Y-STR multiplexes, followed by horizontal non-denaturing polyacrylamide gelelec-trophoresis in 101 unrelated males of Tibetan ethnic group in Lasa of China.A total of 31 different haplotypes were found, 16 of them being unique. The haplotype diversity value (which is the same as the discrimination index) calculated from all three loci combined was 0.9481, which is informative.The Y-STR multiplexes provide useful information for forensic analysis and paternity tests and can also be of great benefit for providing information not normally available from autosomal DNA systems.