ObjectiveTo establish a rapid method for evaluating the heterozygosity of Murraya paniculata germplasm materials and provide as a foundation for developing germplasm breeding and innovation measures for M. paniculata. MethodSingle nucleotide polymorphisms （SNPs） were screened from the genome resequencing data of 65 plants of M. paniculata. A self-written script was used to transform 20 SNPs into restriction fragment length polymorphism （RFLP） markers. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） was employed to detect the 20 RFLP markers in 12 M. paniculata germplasm accessions， and the heterozygosity of M. paniculata germplasm accessions was calculated based on the number of enzyme-cutting bands at the 20 RFLP marker sites. Plink was used to calculate the whole genome heterozygosity of 12 M. paniculata germplasm accessions， and the results obtained with different methods were compared. ResultThere was no significant difference in the heterozygosity calculated by the PCR-RFLP method and the genome resequencing method. The PCR-RFLP and genome resequencing methods identified 8 and 9 germplasm accessions， respectively， with a heterozygosity level less than 30%. Seven germplasm accessions with heterozygosity less than 30.00% were calculated by both methods. ConclusionThe PCR-RFLP method established in this study for evaluating the heterozygosity of M. paniculata germplasm demonstrates the precision of 87.5% and the accuracy of 77.8%. This method serves as a reference for developing heterozygosity evaluation methods in other medicinal plant germplasm resources.

ObjectiveTo establish a rapid screening method for germplasm materials of Gastrodia elata with high purity， and lay a foundation for pure line breeding and cross breeding. MethodBased on the whole genome sequencing and population resequencing of G. elata， 20 restriction fragment length polymorphism (RFLP) markers were developed by single nucleotide polymorphism (SNP) sites. The polymerase chain reaction (PCR)-RFLP method was used to carry out restriction endonuclease experiments on 20 RFLP markers of 15 G. elata germplasms. According to the number of enzymatic bands at 20 RFLP marker sites, the purity of 15 germplasms was calculated and evaluated. On this basis, genome resequencing technology was used to verify the assessment results. ResultTen germplasm materials with purity greater than 95% were screened out by PCR-RFLP method， 3 of which had 95% purity and 7 had 100% purity. Nine germplasm materials with purity greater than 95% were screened out by genome resequencing methods, and 8 of them were consistent with the results of PCR-RFLP. ConclusionThe PCR-RFLP method established in this study for screening G. elata germplasms with high purity precision of RFLP markers has 80% precision and 89% accuracy. The method is simple， efficient， and significantly less expensive than genome resequencing method, which provides technical support for pure line breeding of G. elata and references for breeding of other Chinese medicinal materials.

ObjectiveUncommon medicinal herbs are valuable medicinal resources， but their identification is a difficult problem in Chinese medicine due to their particularity and complexity. It is， therefore， urgent to establish a method for the identification of uncommon medicinal herbs. In this study， DNA signature sequence （DSS） tags were used to establish a specific polymerase chain reaction （PCR） identification method for Hibisci Cortex， the origin plant of Hibisci Cortex， and its adulterants. MethodThe candidate DSS tags were obtained from the chloroplast genome sequence analysis， and the DSS tags were verified by DNA sequencing. The specific identification primers for H. syriacus were designed based on the obtained reliable DSS tags. The PCR reaction conditions were optimized， and the tolerance and feasibility were investigated. ResultA DSS tag for identification of H. syriacus was obtained from the comparison of sequencing results of the amplified products with DSS， which revealed the distinguishing characteristics of Hibisci Cortex and its adulterants. A pair of specific primers for H. syriacus was designed according to the DSS tag. After PCR amplification and gel electrophoresis with the primers， a single bright band of about 270 bp was observed from H. syriacus， which did not appear in the four adulterants. ConclusionA DSS tag obtained in this study can be used to identify H. syriacus. The specific primers designed based on this DSS tag can accurately and simply identify the original plant of Hibisci Cortex and its adulterants， which provides a new method and idea for the molecular identification of genuine and counterfeit products of Hibisci Cortex.

Objective To explore the distribution of genetic structure of Y-SNP and Y-STR genetic markers in different ethnic groups and its application in forensic science. Methods SNaPshot minisequencing was used to detect the polymorphisms of 12 Y-SNP loci in 439 males from 6 ethnic groups, including Guangxi Han, Guangxi Jing, Guangxi Miao, Guangxi Yao, Guangxi Zhuang and Guangxi Dong. DNATyperTM Y26 kit was used to multiplex-amplify 26 Y-STR loci. The PCR products were analyzed by 3130xl genetic analyzer. The network analysis of Y-STR haplotype under the same Y-SNP haplogroup was analyzed by Network 5.0 software. Results Six haplogroups defined by 12 Y-SNP loci were detected in 6 ethnic groups, and 362 haplotypes were detected in 26 Y-STR loci. The haplotype diversity was 0.996 6. In the C haplogroup, the samples from Guangxi Yao, Guangxi Zhuang and Guangxi Dong were clustered on different branches; in the O1 haplogroup, those from Guangxi Zhuang, Guangxi Miao and Guangxi Jing were relatively independent and clustered separately; in the O2 haplogroup, some samples from Guangxi Miao and Guangxi Yao were gathered in a cluster. Conclusion Based on the Y-STR network analysis of samples with identical haplogroup of Y-SNP, some ethnic groups can be preliminarily distinguished, which could be used to infer male suspects' ethnic group through detecting their genetic markers left in the crime scene.
China ; Chromosomes, Human, Y ; Ethnicity ; Genetics, Population ; Haplotypes ; Humans ; Male ; Microsatellite Repeats

A new method for detection of Escherichia coli exist in licorice decoction was developed by using DNA-based electrochemical biosensor. The thiolated capture probe was immobilized on a gold electrode at first. Then the aptamer for Escherichia coli was combined with the capture probe by hybridization. Due to the stronger interaction between the aptamer and the E. coli, the aptamer can dissociate from the capture probe in the presence of E. coli in licorice decoction. The biotinylated detection probe was hybridized with the single-strand capture probe. As a result, the electrochemical response to Escherichia coli can be measured by using differential pulse voltammetric in the presence of α-naphthyl phosphate. The plot of peak current vs. the logarithm of concentration in the range from 2.7×10² to 2.7×10⁸ CFU·mL⁻¹ displayed a linear relationship with a detection limit of 50 CFU·mL⁻¹. The relative standard deviation of 3 successive scans was 2.5%，2.1%，4.6% for 2×10²，2×10⁴，2×106：⁶ CFU·mL⁻¹ E. coli, respectively. The proposed procedure showed better specificity to E. coli in comparison to Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis. In the detection of the real extractum glycyrrhizae, the results between the proposed strategy and the GB assay showed high degree of agreement, demonstrating the designed biosensor could be utilized as a powerful tool for microbial examination for traditional Chinese medicine.

Cell culture is an important tool for biological research.To better understand the pathogenesis and therapeutic methods of the tumors,the three-dimensional cell culture is applied by more and more researchers to create a culture environment that closes to the tumor microoenvironment.Thanks to the advances in the tissue engineering technology,many kinds of models of the three-dimensional cell culture achieve wide accessibility.Compared with the traditional two-dimensional cell culture,the three-dimensional cell culture is better in simulating physiological features of the human histology and cells,including cell proliferation and differentiation,the interaction of cell to cell and cell to matrix.This paper reviews the progress of multi-cellular tumor spheroids (MCTS) culture technique of the three-dimensional cell culture for treatment of bladder cancer.

The optimum harvest time of Tulipa edulis was explored based on biomass accumulation and medicinal quality evaluation. Samples were taken from bud stage (Feb 13th) to dormancy stage (May 14th) and the growth indexes, organs biomasses, drying rate, contents of water-soluble extract and polysaccharides were determined. The results showed that biomass distribution of T. edulis varied with growth center and the bulb gained maximum biomass allocation in the whole growth period. The total biomass accumulation and bulb biomass accumulation　increased in the whole growth period and peaked in fructescence stage. No differences were observed in bulb biomass among fructescence stage, withering stage and dormancy stage. The correlation between bulb biomass allocation and other morphological indexes varied with the harvest time. Bulb dry weight biomass had negative correlation with some morphological indexes of aerial part of T. edulis at bud stage, flower stage and fructescence and had significant positive (P<0.05) or extremely significant positive correlation（P<0.01）with other morphological indexes except for root at bearing fruits stage. The drying rate of bulb of T. edulis increased with the extension of harvest time and peaked in dormancy stage. The water-soluble extract of T. edulis bulb was the highest in pre-growing-stage. The tendency of polysaccharides contents showed a W-shape variation during the harvesting period. The polysaccharides content was the lowest in fructescence stage and was the highest in dormancy stage. Considering the yield and medicinal quality of T. edulis bulb, the optimum harvest time of T. edulis is in the withering stage or early stage of dormancy.

OBJECTIVETo induce Mycobacterium tuberculosis (MTB) resistance with ofloxacin (Ofx) of stepwise increasing concentration in vitro, investigate stability to fluoroquinolone (FQs) antibiotic of MTB, and analyze the molecular mechanism and mutation specialty of drug resistance preliminarily.

METHODSMTB Standard strain H37RV and 24 clinical isolates susceptible to Ofx were selected and experimentally serially subcultured in liquid culture medium containing increasing concentration of Ofx and induced the drug resistance to Ofx. Variety of Minimal Inhibitory Concentrations (MICs) to FQs drugs were detected by microwell-MIC-test method. Mutations of quinolone resistance determining region (QRDR) of gyrA gene were sequenced and identified. Relationship of different mutation sites and drug resistant degree were analyzed. A total of 6 MTB clinical isolates resistant to Ofx and induced drug resistant isolates in vitro were serially subcultured in liquid culture medium without drug. Variety of drug resistant stability, including MIC and mutation of gyrA gene were detected.

RESULTSMIC values of 21 Ofx susceptible isolates after induction were eight times higher than before, which were induced to drug resistant strains successfully and also resistant to Lfx and Mfx. Hot mutations of QRDR of gyrA gene were detected by sequencing, except one strain. Mutation of codon 94 occurred in 60% (12/20) of the strains with mutations and corresponding value of 50% Minimal Inhibitory Concentrations(MIC50) was ≥ 8 µg/ml. In all, 4 of 6 MTB clinical isolates resistant to Ofx harbored mutation of codon 90 (67%) , but the corresponding value of MIC50 was 2 µg/ml. After 21 serially subcultured in liquid culture medium without drug, MIC values of 6 clinical isolates resistant to Ofx were not changed obviously and mutations were also not changed. After 11 times serially subcultured in culture medium without drug, MIC values of induced drug resistant strains were also not changed obviously, but new mutations were detected in QRDR of 3 isolates.

CONCLUSIONMTB strains resistant to three kinds of FQs antibiotic were obtained by induction in vitro with Ofx. Codons 88, 94 mutations of QRDR of gyrA gene were related to the high level FQs drug resistance of MTB. Drug resistant stability of MTB to FQs was strong, and it is difficult for MTB to resume susceptibility.

Antitubercular Agents ; pharmacology ; DNA Gyrase ; genetics ; Drug Resistance, Bacterial ; genetics ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Ofloxacin ; pharmacology

OBJECTIVETo obtain the C7C peptide ligands of Mycobacterium tuberculosis by affinity screening based on the phage-displayed random C7C peptide library, and preliminarily identify the binding capacity of the peptide to Mycobacterium.

METHODSInactive Mycobacterium tuberculosis reference strain H37Rv was used as the target molecule to screen the Ph. D.-C7C peptide library, and Mycobacterium bovis, BCG was used for reverse screening. After 4 rounds of affinity screening, single phages eluted by H37Rv and BCG were selected for DNA sequencing. ELISA was used to detect the binding affinities of different single phage clones. The cyclic peptides displayed by the phage clones showing the highest appetency were synthesized in vitro with fluorescent markers. Fluorescence microscopy and flow cytometer was used to detect the binding affinities of synthesized cyclic peptides, comparing with linear binding peptides obtained before.

RESULTSAfter 4 rounds of biopanning, phages that could bind with target molecules were remarkable enriched. 16 common sequences were obtained by sequencing analysis of single phages. With ELISA, phage SB1, SB5, SB8 and SB26 all showed higher affinity with H37Rv and BCG, the ratio to negative control of which were ≥ 2.1, but could not bind to the 3 nonmycobacteria, which were identified as the positive clones. Based on the results of flow cytometer detection, the affinities to H37Rv of 4 cyclic peptides SB1, SB5, SB24, SB26 were (73.2 ± 6.3)%, (63.2 ± 5.3)%, (32.9 ± 3.1)%, (89.4 ± 7.0)%, and to BCG were (65.6 ± 6.1)%, (48.6 ± 4.5)%, (10.3 ± 1.8)%, (86.6 ± 7.9)%, separately, which were all higher than H8 ((4.0 ± 1.0)%, (5.5 ± 1.2)%) . From the results of fluorescence microscopy observation, all of the fluorescent labeled cyclic peptides SB1, SB5, SB24, SB26 could bind to H37Rv and showed higher fluorescence intensities, which also had certain affinities to other 18 mycobacteria, but the fluorescence intensities were lower than H37Rv, and didn't bind to 3 non-mycobacteria.

CONCLUSIONBased on the replacement of linear 7 peptide library with C7C peptide library, new ligands of Mycobacterium tuberculosis were achieved successfully, which showed significantly higher binding affinities to mycobacteria.

Base Sequence ; Enzyme-Linked Immunosorbent Assay ; Ligands ; Mycobacterium tuberculosis ; Peptide Library ; Peptides

Objective To construct the recombinant plasmid of protein CFP10-MPT48-TB8.4 of Mycobacterium tuberculosis and to investigate the diagnosis potential of this fusion protein in tuberculosis serodiagnosis.Methods The recombinant fusion protein CFP10-MPT48-TB8.4 was expressed, and identified by Western blot.The ELSIA based on the purified fusion protein was done,and used for screening in 230 cases of clinical serum samples including pulmonary tuberculosis patients ( n =150 ),pulmonary disease patients other than tuberculosis (n =70) and health controls (n =103 ).The test result was analyzed by Medcale11.5 software.Results The fusion protein CFP10-MPT48-TB8.4 was successfully expressed with a purity over 95％.Specific immunogenicity of the recombinant protein was confirmed by Western blot.The overall sensitivity and specificity obtained of ELISA were 56.7％ (85/150) and 90.8％ ( 157/173 ),respectively.The specificity was 85.7 ％ (60/70) in non-tuberculosis group and 94.2％ (97/103 ) in healthy group,respectively.Conclusion The recombinant protein of CFP10-MPT48-TB8.4 has a high sensitivity and specificity and may be a potential candidate antigen in tuberculosis serodiagnosis.