Application of rapid PCR to authenticate Ranae Oviductus / 中国中药杂志

Rapid allele-specific PCR primer was designed base on Cytb 155 A/T single nucleotide polymorphism, DNA was extracted by alkaline lysis and the PCR reaction systems including denatured and annealing temperature and cycle numbers were optimized. The results were performed to authenticate Ranae Oviductus and its 4 adulterants. When 100×SYBR Green I was added in the PCR product at 90 ℃ denatured 3 s, 62 ℃ annealing 20 s and 32 cycle. Ranae Oviductus visualized strong green fluorescence under 365 nm UV lamp whereas adulterants appeared negative. The whole process can be completed in 40 minutes．The established method provides the technical support for authentication of the Ranae Oviductus.

Comparative study of PCR effect of rapid PCR instrument and common PCR instrument / 中国法医学杂志

Objective To compare the PCR effect of rapid PCR instrument and common PCR instrument. Methods The concentration of 9947A standard was diluted to 0.1, 0.05, 0.025, 0.0125, 0.0063 ng/μL, 100 samples from each concentration group to establish PCR reaction system with Identifiler? Plus PCR Amplification Kit, 50 samples of them test with rapid PCR instrument (Speed cycler2 thermocycler), the other 50 samples test with common PCR instrument (9700 thermocycler). Detection of PCR product with 3500xL Genetic Analyser, the STR typing of both groups of each concentration group should be compared. Results The success rate of both thermocyclers have no significant difference (P>0.05); The success number of STR typing of common PCR instrument (13.7±1.0; 11.3±1.5) were higher than rapid PCR instrument (13.1±1.3; 9.9±1.9) when the concentration of 9947A were 0.0125, 0.0063ng/μL (P=0.029; P<0.001); The peak height of DNA typing map obtained from common PCR instrument (18931±4625;13437±3165; 5752±1344) were higher than rapid PCR instrument (16929±4034; 11815±4120; 4865±1401) when the concentration of 9947A were 0.1, 0.05, 0.025ng/μL (P=0.023; P=0.030; P=0.002). Conclusions The rapid PCR instrument could achieve the equal success rate to the common PCR instrument with less time, which revealed that the rapid PCR instrument was suitable for application in practical cases; The quality of STR typing from common PCR instrument may be more higher.

Authentication of Cuscutae Semen, Raphani Semen and their adulterants by rapid PCR / 中国中药杂志

To establish an accurate, rapid and efficient method for authenticating Cuscutae Semen and Raphani Semen by using rapid PCR amplification. The samples of Cuscutae Semen, Raphani Semen and their adulterants were collected. The total DNA of the samples has been extracted, and ITS sequence from Cuscutae Semen, Raphani Semen and their adulterants was amplified by PCR and sequenced directionally. These sequences were aligned by using Clustal W. Specific primers were designed and amplified by two-steps PCR amplification method. The rapid PCR methods for authenticating Cuscutae Semen and Raphani Semen were established by optimizing the denatured and annealing temperature, cycle numbers, and etc. When 100 × SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV lamp whereas adulterants showed no florescence. The results indicated that the rapid PCR method can identify Cuscutae Semen and Raphani Semen rapidly. This study provides the technical support for authentication of Chinese medicinal materials.

Establishment and Verification of 6-color Fluorescent-labeled Rapid PCR Amplifi-cation System / 法医学杂志

Objective To establish the rapid PCR am plification program and system and to verify the technical indexes. Methods PCR m ultiplex and capillary electrophoresis detection of 24 autosom al STR loci and one Y-STR loci using the 6-color fluorescence m arking technology, as w ell as Amelogenin and Y-InDel. Meanw hile, sensitivity, specificity, identity, stability, m ixing and a batch of sam ple tests w ere investigated, and the genotype of various routine sam ples and degraded, exfoliated cell sam ples w ere observed. Results The sensitivity of the system w as 0.062 5 ng. In addition, the genotype could be detected accu-rately only around 65 m in via rapid am plification. The species-specificity w as high and the genotyping of all kinds of dry blood specim ens of filter paper and m ixed, degraded, exfoliated cell sam ples w ere accu-rate. Conclusion The rapid am plification system can significantly im prove the detection rate, and obtain accurate and stable genotyping results, w hich m ay be im portant im plications for the establishm ent of STR database and study on population genetics and forensic identification.

Rapid molecular identification of saffron (Crocus sativus L.) based on specific PCR / 中国药学杂志

OBJECTIVE: To establish a rapid molecular method for identifying saffron (Crocus sativus L.) and its adulterants by PCR: amplification using specific primers and fluorescent dyes detection. METHODS: The chloroplast barcode was sequenced and analyzed to find the SNPs between saffron and its adulterants. Specific primers were designed for the SNPs, the PCR reaction systems were built and optimized, and fluorescent dyes method was used to identify PCR products. RESULTS: A 421 bp saffron identification band based on the psbA-trnH barcode sequence was screened when 100 × SYBR Green I was added into the optimized PCR product under the following condition; initial denaturation at 90℃ for 1 min, denaturation at 90℃ for 5 s, annealing at 58℃ for 5 s, 26 cycles; the saffron (Crocus sativus L.) showed strong green fluorescence under 365 nm UV lamp whereas adulterants did not. CONCLUSION: Fast site-specific PCR can rapidly identify saffron and its adulteration.

The Development of Rapid PCR / 中国生物工程杂志

Polymerase chain reaction (PCR) is one of the common techniques in molecular biology, which can amplify nucleic acids through the cycle of denaturation, annealing and extension. Based on the principle of common PCR, rapid PCR is to realize the amplification of nucleic acids in less time without affecting the specificity, sensitivity and fidelity of the reaction. A lot of research work in this field has been going on in recent years. This article will make a review of the development of rapid PCR with emphases on the improvement of DNA polymerase, the choice of additives and the improvement of thermocyclers.