High resolution melting (HRM), based on melting curve analysis, requires not only saturating dyes that fluoresce in the presence of double-stranded DNA, but also higher resolution detection equipment. The melting curve is a novel method for sequence matching, genotyping and mutation scanning. The technology is simple, accurate, rapid, closed-tube, low-cost, and high-throughput, which make it gain more and more applications. This review article presents the basic principles, key factors and both the advantage and limitations of HRM. The potential application is discussed in the study of molecular identity of traditional Chinese medicine.
DNA Mutational Analysis ; methods ; Drugs, Chinese Herbal ; classification ; Genotyping Techniques ; methods ; Medicine, Chinese Traditional ; Nucleic Acid Denaturation

High resolution melting (HRM) , an important technology for genotyping and mutation scanning, has broad prospects in the authenticity of traditional Chinese medicine. This paper selected universal CO I primers and used HRM to establish a new method for authenticity of Hairy Antler. PCR was conducted at the annealing temperature of 60 °C and 45 cycles. The range of the DNA template concentration, the primer concentration and the Mg2+ ion concentration were further optimized. The results showed that the Tm values of Cervus nippon were (81.96 ± 0.07), (84.51 ± 0.03) °C and Cervus elaphus was(82.58 ± 0.13), (85.95 ± 0.05) °C with 10-100 mg · L(-1) DNA template, 0.2 µLmol · L(-1) primer, 2.0 mmol · L(-1) Mg2+. This method can authenticate of hairy antler and is simple, fast, high-throughput, visualization.
Animals ; Antlers ; chemistry ; DNA ; chemistry ; genetics ; DNA Primers ; genetics ; Deer ; classification ; genetics ; Genotype ; Medicine, Chinese Traditional ; standards ; Polymerase Chain Reaction ; Transition Temperature

A total of 12 775 SSRs were identified from Scutellaria baicalensis Georgi genomic database, accounting for 2.56% of the total genomic sequences. The result showed that S. baicalensis SSRs were based on 68.32% dinucleotide and 18.63% trinucleotide repeats; CT/GA and TTC/GAA were predominant in the dinucleotide motifs and the trinucleotide motifs respectively. Nine primers were selected to produce highly reproducible SSR bands and were used in studying the genetic diversity of S. baicalensis, 50 individuals from ten populations. 68 SSR polymorphic loci were detected, these loci were polymorphic and displayed 4 to 12 alleles per locus with a mean number of 7; the effect number of alleles was 3. Expected heterozygosities were 0.6 and were far more greater than the average in dicotyledonous plants. PIC (polymorphism information content) was 0.72, Shannon's information index was 1.32, these all proved that S. baicalensis had a high genetic diversity in general. Genetic differentiation among population Gst was 0.131, genetic variation among population accounted for 13.1% and genetic variation within population accounted for 86.9%. The cluster analysis showed that 10 populations S. Baicalensis were classified into 2 groups, but it was not associated with geographical distribution.
Alleles ; Cluster Analysis ; Genetic Variation ; Genomics ; Microsatellite Repeats ; Scutellaria baicalensis ; genetics ; Trinucleotide Repeats

In this paper, Liuwei Dihuang pill was used to study the identification of Chinese patent medicine by fluorescence sequencing typing technology. The DNA of Paeonia suffruticosa was used as template to amplify by five pair of FAM fluorescence labeling primers. Then, the amplified products were sequenced. The sequencing results were analyzed by GeneMarker V1.80 to screen the best fluorescence labeling primers. As a result, psbA-trnH fluorescence labeling primer was used to identify the raw materials of Liuwei Dihuang pill. The results showed that three kinds of raw plant medicinal materials in Liuwei Dihuang pill were able to be correctly identified by psbA-trnH fluorescence labeling primer. The fluorescence sequencing typing technology can stably and accurately distinguish raw medicinal materials in Chinese patent medicine.
DNA Primers ; chemistry ; genetics ; DNA, Plant ; chemistry ; genetics ; Drugs, Chinese Herbal ; chemistry ; standards ; Fluorescent Dyes ; chemistry ; Plants, Medicinal ; chemistry ; genetics ; Polymerase Chain Reaction ; instrumentation ; methods ; Quality Control ; Staining and Labeling

To obtained an accurate, rapid and efficient method for authenticate medicinal snakes listed in Chinese Pharmacopoeia (Zaocysd humnades, Bungarus multicinctus, Agkistrodon acutus), a rapid PCR method for authenticate snakes and its adulterants was established based on the classic molecular authentication methods. DNA was extracted by alkaline lysis and the specific primers were amplified by two-steps PCR amplification method. The denatured and annealing temperature and cycle numbers were optimized. When 100 x SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV whereas adulterants without. The whole process can complete in 30-45 minutes. The established method provides the technical support for authentication of the snakes on field.
Animals ; China ; DNA Primers ; genetics ; Polymerase Chain Reaction ; methods ; Reptilian Proteins ; genetics ; Snakes ; classification ; genetics

To simply and rapid authenticate Lonicera japanica. Rapid allele-specific PCR primer was designed base on trnL-trnF 625 G/T Single nucleotide polymorphism and the PCR reaction systems including annealing temperature was optimized; optimized results were performed to authenticate L. japanica and its 9 adulterants. When 100 x SYBR Green I was added in the PCR product of 87 degrees C initial denatured 1 min; 87 degrees C denatured 5 s, 68 degrees C annealing 5 s, 30 cycle; L. japanica visualize strong green fluorescence under 365 nm UV lamp whereas adulterants without. The results indicate rapid allele-specific PCR could authenticate L. japanica and its adulterants rapidly and simply.
Alleles ; DNA Primers ; genetics ; Drug Contamination ; prevention & control ; Lonicera ; classification ; genetics ; Polymerase Chain Reaction ; methods ; Quality Control

Objective:To study the collapse of the contralateral femoral head after the total hip arthroplasty (THA) in patients with bilateral steroid-induced femoral head necrosis leg-length discrepancy (LLD) influences.Methods:A total of 108 patients with bilateral steroid-induced femoral head necrosis who were treated in Honghui Hospital Affiliated to Xi′an Jiaotong University from June 2014 to June 2016 underwent THA surgery on the hip joints that had symptoms and developed to Association Research Circulation Osseous (ARCO) Ⅲ. At the same time, the non-surgical hip-preserving treatment of the non-collapsed femoral head developed to the ARCO Ⅱ stage was performed. The follow-up period was 2 years, and 98 cases were finally included. According to the size of the leg-length discrepancy [LLD<3 mm group ( n=50), LLD≥3 mm group ( n=48)] and the type of leg-length discrepancy[non collapse side longer group ( n=58) and shorter group ( n=58)], the collapse of the femoral head and the THA were observed. Results:Finally, a total of 56 cases of femoral head collapse occurred in the non collapse side of the hip, of which 50 cases underwent THA within 24 months. There were significant statistical differences in THA and femoral head collapse between LLD<3 mm group and LLD≥3 mm group, non collapse side longer group and non collapse side shorter group ( P<0.05). The 2-year survival rate without collapse in the LLD<3 mm group and LLD≥3 mm group were 52.1% and 34.0%, respectively, and the 2-year survival rates in the longer and shorter non-collapsed limb groups were 56.9% and 22.5%, respectively. Compared with patients with moderate lesions, the survival rate of femoral heads in patients with larger lesions was lower ( OR: 4.25, 95% CI: 1.55-11.26; P=0.003). LLD<3 mm group ( OR: 0.24, 95% CI: 0.06-0.50; P<0.01) or non collapse side longer group ( OR: 0.13, 95% CI: 0.04-0.29; P<0.01) had lower risk of contralateral femoral head collapse after THA. Conclusions:For patients with bilateral steroid-induced femoral head necrosis who have collapsed lateral THA, postoperative leg-length discrepancy extremities is a potential risk factor for collapse of non-collapsed femoral head. LLD<3 mm and avoiding shortening of the uncollapsed limb may reduce the risk of collapse of the uncollapsed femoral head.

Molecular identification of Chinese traditional medicine has come from laboratory research into application, but there are some misunderstandings and problems emerging after rapid development. In this paper, we discuss the usage principle, hot field and technology innovation in molecular identification of Chinese traditional medicine. And molecular identification of traditional Chinese medicine has scientific and objective basis, follows the certain systematic research background, and adopts practical principles to establish case by case multi-class identification system. In order to achieve rapid, on-site, high throughput, low cost of traditional Chinese medicine identification purpose, molecular identification technology is further developing for meet the actual needs and the laboratory results further transformation in the service of traditional Chinese medicine industry.
China ; Drugs, Chinese Herbal ; chemistry ; standards ; Medicine, Chinese Traditional ; Plants, Medicinal ; chemistry ; classification ; genetics ; Quality Control

Objective:To test the expression of Minichromosome maintenance complex component 7(MCM7) protein in hepato-cellular carcinoma(HCC) of different species including human, rat and tree shrew (tupaia) by cross-species oncogenomics approach, and to investigate the relationship between the expression of MCM7 and the development of hepatocellular carcinoma and its clinical significance. Methods:Western blot and Immunohistochemistry were applied to detect the expression levels of MCM7 protein in HCC tissues,corresponding HCC-adjacent liver tissues and normal liver tissues collected from different species including human, rat and tree shrew, respectively. The clinicopathologic factors were also analyzed with the results of Immunohistochemistry. Results:Western blot analysis showed that the expression of MCM7 protein in HCC tissues of human and rat were higher than that in corresponding HCC-ad-jacent liver tissues and normal liver tissues, respectively and significantly (P<0.05). However, the expression of MCM7 protein in HCC tissues of tree shrew were also higher than that in corresponding HCC-adjacent liver tissues and normal liver tissues, but no significant difference was found among three types of tissues (P>0.05).There was also no significant difference between HCC-adjacent liver tis-sues and normal liver tissues in three species (P>0.05). Immunohistochemical analysis showed that MCM7 protein was mainly ex-pressed in nucleus of HCC cells, and the positive rate of MCM7 protein in HCC tissues of human, rat and tree shrew were significantly higher than that in corresponding HCC-adjacent liver tissues and normal liver tissues, respectively (P<0.05). However, no significant difference was found between HCC-adjacent liver tissues and normal liver tissues (P>0.05). Moreover, the protein level of MCM7 was intimately related to patient's HCC stage, extrahepatic metastases and postoperative recurrence (P<0.05). Conclusion:MCM7 protein might play a pivotal role in hepatocarcinogenesis. In addition, it was probably related to patient's HCC stage, extrahepatic metastases and postoperative recurrence. It seems very likely that MCM7 may be applied as a new molecular target in HCC prevention and treat-ment.

Objective To investigate the attentional network in patients with temporal lobe epilepsy(TLE) and the disease-related factors. Methods Fity-four patients with TLE and 40 controls were enrolled in our hospital from January 2007 to December 2008.The computerized ANT software was used for evaluating the attentional network efficiency and the clinical date of the patients with TLE were recorded. Logistic regression was used to identify the risk factors of attentional network efficiency. Results The mean reaction time(TLE:688.2±138.1 ms;Control:625.1±100.1 ms, t=2.06, P <0.05)and executive control network efficiency (TLE:155.7±57.0 ms;Control:108.0±33.8 ms, t=4.62, P <0.01) of the TLE group were lower than the healthy control group. The efficiency of alerting network and orienting network between the two groups were no significant difference. Logistic regression analysis showed that the positive epileptiform activity was an independent risk factor of the attentional impairment(95% CI : 1.03～42.33, OR =6.603, P =0.043). Conclusions The ANT demonstrate that patients with TLE may accompany with attentional executive control network efficiency impairment. Epileptiform discharge may cause attentional executive network efficiency impairment.