With the rapid development of Next-generation Sequencing(NGS)technologies, and its high throughput and low cost is applied widely in the field of life science, the increase in the depth of sequencing together decrease in the consumption of time and cost, makes a wide application of NGS in the research of microbiological research, ancient DNA study, clinical diagnosis, forensic science research, etc. The article discusses the second generation sequencing technology platform and its genetic markers in the forensic application. Included STR typing, SNP typing, HLA genotype prediction and the application in the degradation of the material.

Objective To disclose the inter-species differences among Papaver somniferum L, Papaver rhoeas L and Cannabis sativa L using AFLP analysis. MethodsThe root, stem, foliage, flower, seed of Papaver somniferum L, foliage of Papaver rhoeas L and Cannabis sativa L were collected respectively. DNA was isolated with AxyPrep Kit, and double-digested by EcoR I and Mse I, then oligonucleotide adapters were ligated. After Pre-amplification, selective amplification was performed using 6 pairs of fluorescent primer. The DNA fragments were analyzed using a CEQ 8000 DNA Fragment Analyzer. Results27 to 46, 5 to 20 and 4 to 31 pieces of amplified products were detected from Papaver somniferum L, Cannabis sativa L and Papaver rhoeas L respectively, and significant difference was observed among these three groups of products. The identical species-specific peaks were identified from root, stem, leaf, flower and seed DNA from the same Papaver somniferum L. ConclusionThe diacritical results from Papaver somniferum L, Papaver rhoeas L and Cannabis sativa L, as well as the identical results of different parts from the same plant demonstrated that AFLP analysis could be used possibly to determine the species origin of unknown plants samples.

In forensic case, the location diagnosis determination of drowning has been one of the most important and dififcult diagnosis. Then diatom test is considered to be a relatively reliable method for the diagnosis of drowning. According to community characteristics inferred from diatoms into the different water region has been credibility. The study of the diatoms in different waters identiifcation is reference to the determination of the body drowned into the water area. In this paper, the research progress of diatom relevant biological characteristics and test methods on review for forensic workers is related to the further research and reference case in practice.

Objective To comparatively evaluate the effects of a recombinant Mtb protein ESAT 6-CFP10 ( rESAT6-CFP10 ) and a purified protein derivative ( PPD ) as skin test reagents in guinea pigs . Methods Guinea pigs were sensitized with different Mycobacteria species .After sensitization , all guinea pigs were intradermally injected with rESAT6-CFP10 and PPD.At 48 h after the injection, the size of ery-thema at injection sites was measured by using a double-blind method .For guinea pigs sensitized with viable Mtb, the size of erythema at injection sites were measured at 24 h after the injection .The positive conversion rates of skin test with rESAT 6-CFP10 and PPD were calculated .Results The results of PPD skin test were positive in all guinea pigs sensitized with viable Mtb , killed Mtb and BCG with erythema diameters of (11.4 ±0.9) mm, (11.8±1.1) mm and (13.2±0.8) mm, respectively.Positive skin test with rESAT6-CFP10 was only observed in guinea pigs infected by viable Mtb-showing erythema diameters of (13.7±5.7) mm. The skin test with rESAT6-CFP10 was negative in guinea pigs sensitized by killed Mtb-and vaccinated by BCG.The skin tests by using rESAT6-CFP10 and PPD were performed on randomly selected guinea pigs at ninth day after infection by Mycobacterium tuberculosis H37Rv.At the 2nd week, totally 24 selected guinea pigs showed positive skin test results with rESAT6-CFP10 (24/24) with erythema diameters of (19.9± 3.0) mm, while only 15 out of 24 had positive PPD skin test with erythema diameters of (6.1±5.5) mm. At the 4th week, all guinea pigs showed positive PPD skin test (3/3) with erythema diameters of (12.7± 2.5) mm.Conclusion The skin test by using recombinant ESAT 6-CFP10 protein can effectively distin-guish viable Mtb infection from BCG vaccination and killed Mtb sensitization , which is a more suitable anti-gen than PPD for the early diagnosis of Mtb infection .

Objective To establish a suitable guinea pig model for evaluating the protective effects of new therapeutic TB vaccines in preclinical study .Methods The guinea pigs were subcutaneously injected with single-cell suspension of multi-drug resistant M.tuberculosis at a dose of 1000 CFU, 0.2 ml per animal.The study was divided into experimentⅠand experimentⅡ.In experimentⅠ, the guinea pigs were given immuno-therapy and/or chemical treatment on day 3 after infection .Four guinea pigs in each group were dissected at weeks 5, 7 and 9 after infection for evaluating lesion scores and histopathology changes of liver , spleen and lung, as well as bacterial load in spleen .In experimentⅡ, the guinea pigs were given immunotherapy and/or chemical treatment with different doses on day 14 after infection .All guinea pigs were dissected at week 5 after infection for evaluating lesion scores of liver , spleen and lung , as well as bacterial load in spleen .Results In experimentⅠ, all of the three treatments including immunotherapy combined with chemotherapy , immunotherapy alone and chemotherapy alone could effectively prevent organ lesion and reduce bacterial load in spleen , which were significantly different from negative control group .The immunotherapy combined with chemotherapy showed better treatment effects than other two treatments .Along with a prolonged period after drug withdrawal , the de-gree of organ lesion in immunotherapy group and chemotherapy group rebounded sharply , but only slightly in im-munotherapy combined with chemotherapy group .In experimentⅡ, animals in all treatment groups showed alle -viated organ lesion and reduced bacterial load in spleen .A relatively better treatment effect was observed in im-munotherapy combined with chemotherapy group .Conclusion The established guinea pig model of M.tubercu-losis infection showed an advantage of good repeatability .It might be used to evaluate the protective effects of new therapeutic vaccines against tuberculosis in preclinical study .

Objective Acquiring genetic information of Y-SNPs and Y-STRs genetic makers from samples with the surname of Kong, which is useful for exploring the correlation between surname and Y chromosome in forensic applications studies.Methods Two multiplex genotyping assays and SNaPshot assay were used to analyze 255 unrelated male blood samples who share the same surname Kong and 330 unrelated male blood samples obtained randomly. 17 Y-STRs were typed for the surname Kong population samples. The software Arlequin 3.5.1.2 and the program Network 4.6.1.1 were used for data statistical analysis.Results 13 haplogroups were observed. The highest haplogroup frequency in the two populations were O3a2c1a-M117 (21.57%, 14.85%). 196 haplotypes in Kong population deifned by 17 Y-STRs locus were obtained and the haplotype diversity was 0.9939. 14-12-25-28-19-15-12-19-12-11-12-22-12-11-14-10-19 is the typical haplotype. Median Joining algorithm and Mismatch Distribution were adopted to analyze the Y-STR haplotye under haplogroup O3-M122, and the result shows that there are two “central star” distribution. Conclusion Combined with Y-SNP and Y-STR analysis showed that the Kong population had experienced complicated exchanges and expansion or continued growth, which has more than one surname origin. Hence, its population genetic structure and historical differences have potential applications in forensic science.

Objective T o detect the genotype of A B O blood group by SN aPshot technology. Methods D N A w ere extracted from the peripheral blood sam ples w ith know n blood groups (obtained by serology) of 107 unrelated individuals in Y unnan. Six SN P loci of the 261th, 297th, 681th, 703th, 802th, and 803th nucleotide positions w ere detected by SN aPshot M ultiplex kit, and relevant genetics param eters w ere cal-culated. Results In 107 blood sam ples, the allele frequencies of types A , B , O A, and O G w ere 0.3551, 0.1682, 0.2300 and 0.2476, respectively, w hile that of types A G and cis A B w ere not detected. T he geno-typing results of A B O blood group w ere consistent w ith that of serologic testing. Conclusion SN aPshot technology can be adapted for genotyping of A B O blood group.

Objective To establish a guinea pig model of latent Mycobacterium tuberculosis infec-tion for evaluating the effects of therapeutic vaccines .Methods Guinea pigs were subcutaneously inocula-ted with 5.0×103 CFU Mtb.The skin test was performed with 0.5μg recombinant ESAT6-CFP10 protein to detect positive conversion rates at different time points .Two weeks after Mtb inoculation , guinea pigs in model group received 5 mg isoniazid treatment ( three times a week for four weeks ) by oral gavage , while those in control group received normal saline .At the sixth week after Mtb infection , guinea pigs with and without isoniazid treatment were dissected for pathology examination .The pathological scores of liver , spleen and lung, as well as bacteria loads in spleen were compared between two groups .The established guinea pig model of latent infection was then validated by testing two reference vaccines ( AEC/BC02 and AEC/BC03 ) . Results Two weeks after Mtb inoculation , all guinea pigs showed positive EC skin test with induration area of (19.9±3.0) mm.Upon four weeks of isoniazid treatment , the guinea pigs in model group showed no pathological changes with zero scores in the examined organs .No bacterium was detected in spleen of ani-mals from model group.However, the total pathological score was 38.8±16.5 and bacteria load in spleen was (5.1±0.3) Log10 CFU with the guinea pigs from control group .Natural recurrence of tuberculosis in model group was observed after drug withdrawal .The total pathological scores were 48.5±23.9 and 51.3± 23.41.The bacterial loads in spleen were (4.5±1.3) and (4.2±1.1) Log10 CFU and bacterial loads in lung were (4.1±1.2) and (3.4±1.3) Log10 CFU respectively as verified with reference vaccines of AEC /BC02 and AEC/BC03.Conclusion Isoniazid treatment inhibited the proliferation of inoculated Mtb in guinea pigs.A guinea pig model of latent Mycobacterium tuberculosis infection is successfully established with an advantage of good repeatability .Therefore, it can be used to evaluate the effects of therapeutic vaccines on latent Mycobacterium tuberculosis infection.

Objective To establish a suitable guinea pig model for evaluating the protective effects of new TB vaccines in BCG prime-boost regimen .Methods Two different immunization strategies by using the recombinant TB vaccine were employed to boost BCG primed guinea pigs in this study .One was for short-term evaluation with 14 weeks interval between prime and boost immunization and another was for long -term evaluation with 54 weeks interval .In the short-term evaluation group , guinea pigs were boosted twice with the recombinant TB vaccine ( AEC/BC02 ) in every two weeks , while guinea pigs in the long-term evaluation group were boosted for three times with two weeks interval between each injection .A negative con-trol group ( NS→NS) and a BCG control group ( BCG→NS) were both set up in two evaluation groups .One week after the last immunization , all guinea pigs were challenged with M.tuberculosis.Six to seven weeks after bacteria challenge , all animals were euthanized and dissected to evaluate lesion scores of liver , spleen and lung, as well as the viable bacterial load in spleen .Results In the short-term evaluation group , the le-sion scores in those boosted with vaccine (3.33±5.00) was lower than that of BCG control group (5.56± 7.27) (P>0.05) and negative control group (47.00±28.11) (P=0.0001).The difference between BCG control group and negative control group in lesion score was also significant .The animals in vaccine boosted group had lower bacterial loads (0.78±1.55 log10 ) in spleen than that in BCG control group (1.06±1.87) (P>0.05) and negative control group (5.47±0.61) (P=0.0003).In the long-term evaluation group, the lesion score in those boosted with vaccine was lower (5.0±7.6) than that in BCG control group (14.4± 13.5) (P=0.0394) and negative control group (56.9±14.1) (P<0.0001).The animals in vaccine boos-ted group (1.00±1.86 log10) had lower bacterial loads in spleen than that in BCG control group (1.46± 1.94) (P>0.05) and negative control group (5.43±0.56) (P=0.01).There was a significant difference in bacterial load between BCG control group and negative group (P=0.0089).Conclusion The results suggest that the interval time between BCG-prime and boost immunization should be properly prolonged in the guinea pig model used for evaluating the protective effects of new TB vaccines in BCG prime -boost regimen .

OBJECTIVETo assess the association of 8 single nucleotide polymorphisms (SNPs) from chromosomes X and 20 with androgenetic alopecia among ethnic Han population from Yunnan province.

METHODSAn eight-SNP co-amplification protocol was developed for the genotyping with a SNaPshot platform. A case-control study was carried out for the 8 SNPs from chromosomes X and 20 in 115 androgenetic alopecia cases and 125 healthy controls. Statistical analysis was conducted with SPSS17.0, Haploview4.2, SHEsis and MDR software.

RESULTSNo association was found between the two groups with regard to the 4 SNPs located on the X chromosome. The genotypic frequencies of rs2180439, rs913063 and rs1160312 were significantly different between the two groups (P < 0.05). The frequency of T allele of rs2180439 was significantly higher in the case group (P < 0.05). The frequencies of A alleles of rs913063 and rs1160312 were significantly higher in the case group (P < 0.05). The haplotypes of C-T-C-G, T-C-C-G and T-T-A-A based on rs6137444-rs2180439-rs913063-rs1160312 showed significant difference between the two groups (P <0.05). rs6137444, rs21804393 and rs1160312 have a strong association with androgenetic alopecia.

CONCLUSIONThe 4 SNPs located on chromosome X were all monomorphic among ethnic Hans from Yunnan. The rs6152, rs16990427, rs1352015, rs1385699 SNPs located on chromosome 20 are associated with androgenetic alopecia in the same population. Individuals with T allele of rs2180439 and A allele of rs913063 and rs1160312 are more likely to develop androgenetic alopecia.

Adult ; Alopecia ; genetics ; Case-Control Studies ; China ; ethnology ; Chromosomes, Human, Pair 20 ; Chromosomes, Human, X ; Genotype ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide