The isleof gene-specific PCR technology identifies The Truth and Falseoft.

Abstract: The purpose is to establish a method to quickly identify the authenticity of Ho.U. The
method, through the psbA-trnH gene sequence of He Shouwu and his mixed counterfeits, looks for the SNP site and designs specific primers, and makes PCR amplification of He Shouwu and its three conjure sprites from different origin, optimizes the reaction system conditions, and investigates this method.
results established the method of He Wu-specific PCR, in the annealing temperature of 48 degrees C, the number of cycles 30, only what first wu energy amplification to get 191 bp of specific strip, counterfeit sufflud no.
Conclusion The isleof-specific PCR method of identifying The Truth of Ho's first wu is simple and reliable.
He Shouwu was published in the Tang Dynasty's "He Shou Wu" , for the clinical commonly used Chinese herbal medicines.
the 2015 edition of the Chinese Pharmacopoeia, respectively, contains He Shouwu and the production of He Shouwu, of which He ShouWuwu Bogum multiumflorThunb's dry root, with anti-venom antimalarial malaria, intestinal stool and other effects.
Because of the uniqueness of the Sect.Tiniaria plant, Flora of China independently of the plants of the cranberry group fallopia Adans, so Ho-Wu's Latin school name is Fallopia Multiflora (Thunb.) Harald.
for a long time, there has been confusion in different regions, especially the source of the drug in the private medicine.
market also often appears in the first Wu-Yuan medicinal spostots, such as the first Wu Fu.multiflora Thunb.var.angulata S.Y. Liu, the hairy f.multiflora (Thunb.) Harald.var.ciliinervis (Nakai) Yonekura and H. Ohashi, toothed leaf f.denticulata (Huang) A.J. Li equidistant species .
traditional Chinese medicine methods are mainly based on the characteristics of medicinal herbs and simple physical and chemical identification methods to identify, but it is difficult to identify some of the morphological and chemical composition of the very similar Chinese herbs, such as He Shouwu and variants of the prismatic branch of He Shouwu, the two are very similar in form and chemical composition, both have a spindle-shaped or clump-like block root, and the root of the block has abnormal maintenance tube beam sexist, traditional methods are difficult to identify.
previous studies have shown that the content of the active ingredient of The First Wu is low and cannot be mixed with Ho-Chi-Wu.
in recent years, with the continuous development and wide application of DNA molecular genetic marker technology, it makes up for the lack of traditional identification in the identification of traditional Chinese medicine, and plays an important auxiliary role.
among the many molecular marker methods, the most widely used is single nucleotide polymorphism (single nucleotide polymorphisms, SNP).
at present, there are also scholars using molecular marking technology for ginseng, three seven, fishy grass, Zhejiang mother and many other medicinal materials authenticity and base original identification.
Li Jiewen was able to identify Ho And his mixed artifacts using allele-specific PCR technology based on ITS2 sequence.
however, because the nuclear gene has the characteristicof of multi-copy, it is easy to have gene overlap when identifying the same plant.
therefore, based on the difference of psbA-trnH sequence of He Shouwu and mixed counterfeits, this study, by introducing the specific prime rinpartation of the dismatching base in the second place of the 3'end-end penultimate, so that he first Wu appears specific band, and the counterfeit strucizing without specific strip identification, the establishment of allele-specific PCR technology to identify Ho Shou Wu authenticity.
1 instruments and materials SimpliAmpTM96-hole PCR (Applied Biosystems Company); MM400 Frozen Mixing Ball Grinder (Retsch Company); GI54TR Vertical Auto-Pressure Steam Sterizer (Micro Xiamen InstrumentCoB); Micro 21R desktop high-speed refrigeration centrifuge (Thermo company); NanoPhotometer NP80 ultra-micro-specvelight photometer (Implen company) ;D YY-6C electrophoremeter (Beijing Six-One Biotech) and Alphalmager HP Gel Imaging System (ProteinSimple).
1.2 Materials Pfu Taq DNA polymerase, r Taq DNA polymerase, DNA Marker, 4S Red Plus (Bioengineering (Shanghai) Co., Ltd.), Ex Taq DNA polymerase (TaKaRa Company);
samples from Anhui, Zhejiang, Henan, Guangxi, Guangdong, Sichuan, Guizhou, Hunan, Hubei, Yunnan and other provinces were collected from a total of 77 materials.
all samples were identified by Professor Peng Huasheng of Anhui University of Traditional Chinese Medicine, and the voucher specimens were stored in the Chinese Medicine Resource Center of Anhui University of Traditional Chinese Medicine.
sample details can be found in Table 1.
2 method 2.1 SNP site screening and specific primer design using BioEdit software to sequence the results and GenBank database of He Shouwu and its equivalent of mixed counterfeitps psbA-trnH sequence to be ho-sourced alignment, manual correction to find differential SNP sites, see Figure 1.
designed by Primer Premier 5.0 software to identify the specific primer FMF-FMR of He Shouwu and its mixed artifacts, the primer synthesized by Bioengineering (Shanghai) Co., Ltd., and the sequence and PCR amplification program is shown in Table 2.
the extraction of the total DNA of the 2.2 genome and the determination of PCR amplification conditions, the total DNA is extracted from the dry sample 25 to 50 mg, placed in an EP tube of 2.0 mL, and the total DNA is extracted according to the improved 2% CTAB method.
take different sample DNA, adjust the mass concentration to 10 to 100 ng/mL, and use the upstream and downstream primers FHS and FHA of the psbA-trnH sequence to detect the quality of the template DNA.
PCR reaction system (total volume of 25?L) consisting: 10 x PCR buffer 2.5?L, 2.5 mmol/L dNTPs 2?L, 25 mmol/L MgCl2 2?L, primer FHS 1?L, primer FHA 1?L, Ex Taq DNA polymerase 0.125L, DNA template 1 sl,
reaction is performed on the SimpliAmpTM 96-well PCR instrument, the reaction conditions are shown in Table 2. After the
reaction, the PCR amplification yield was taken by 3 sL, 2 sL 6 x load buffer, mixed in 4S Red Plu dyed 1% agaulcane gel electrophoretic detection, Alphalmager HP gel imaging system observation imaging.
2.3 He Shouwu and his con-mixed pseudo-grade point-specific PCR identification using FMF-FMR-specific primers for specific PCR reactions to different samples, PCR reaction system (total volume of 25 ?L) composition: 10 x PCR buffer 2.5 sL, 2.5 mmol/LNTPs 0.5 L, 25 mmol/L MgCl2 1.5?L, primer FMF 1?L, primer FMR 1?L, r Taq DNA polymerase 0.5?L, DNA template (10 to 100 ng) 1 ?L, sterile distilled water to 25 sL.
reaction conditions can be found in Table 2.
with 2.5% agarose gel electrophorecin detection specific PCR expansion, in 100 V voltage conditions electrophoresis about 30 min, and then use gel imaging analyzer to take pictures, if the electrophoresis results appear 191 bp DNA strip, then why the sample to be tested, if the electrophoresis map does not appear 191 bp DNA strip, the sample to be measured is why the first counterfeit.
the effects of annealing temperature, PCR cycle number, primer concentration, Taq enzyme type, MgCl2 dosage on the results of identification of He Shouwu and his mixed artifacts, respectively, to determine the optimal reaction system and conditions.
3 results and analysis of 3.1 allele gene specific PCR primer design of He Shouwu and his equivalent of 77 samples of mixed counterfeits of the psbA-trnH sequence analysis showed that psbA-trnH existed 3 SNP sites.
165th base is T, 236th base is A and 311st is C Why the first Ute has the SNP site.
use these three SNP sites, design Ho Shou wu and the same mixed counterfeit seile gene-specific PCR primer, set the positive and reverse primer of the 3' end and the SNP site exactly match, and the 3' end of the penultimate place introduced to the mismatch editing base.
used the design's primers to amplify He Shouwu and his conjure samplification with A special strip length of 191 bp.
3.2 Allegene-specific PCR amplification reaction conditions optimized at the same instrument for the optimization of isotron-specific PCR amplification reaction conditions for the annealing temperature, number of cycles, primer concentration, dosage of MgCl2 and different different taq enzymes at the same time to determine the optimal reaction conditions.
this experiment examined eight annealing temperatures, 42, 44, 46, 48, 50, 52, 54, 56 degrees C, and found that the annealing temperature in 42 to 56 degrees C only the first upped out of the special strip, counterfeits are not.
however, when the annealing temperature is 42 to 46 degrees C and 56 degrees C, nonspecific strips can be seen, see Figure 2-A; No, the number of cycles is less than 24 hours, genuine and counterfeit are no strip, see Figure 2-B;
when FMF/FMR is 1 sL, Ho's strip is the brightest, see Figure 2-C;
this may be related to the high-fidelity enzyme can correct the base of the 3'-5' extyl enzyme mismatch, resulting in the experimental specific primer 3' end dispensing of the base is enzyme-cut, therefore, the amplification performance is consistent with conventional PCR.
, in addition, the mgCl2 dosage, respectively, 0.5, 1.0, 1.5, 2.0, 2.5?L, it was found that the mgCl2 dosage is less than 1.0?L, he and the counterfeits are not specific strip, Only more than 1.0 ?L, He Shouwu has the opposite sex strip, the counterfeit is none, see Figure 2-E, in view of the mgCl2 dosage on the identification of He Shouwu has little impact and for the experimental cost considerations, so choose MgCl2 dosage for 1.5 sL.
3.3 accuracy test using optimized allele-specific PCR reaction conditions, the ilegen-specific PCR amplification of 77 samples, the results only appeared in the first 191 bp-specific band, the other 3 same-type conjures were non-false positive and false negative results.
partial results are shown in Figure 3.
3.4 detection limit sit u.s specific PCR for template DNA of different mass concentrations according to optimized PCR reaction conditions.
THE QUALITY CONCENTRATION OF THE DNA TEMPLATE IS 10 to 100 NG/mL, only he can amplify 191 bp of specific bands, and the brightness of the strip decreases with the dna template concentration decreases, the template DNA detection limit is up to 10 ng/mL, see Figure 4.
4 discuss the distribution of the genus Ho first in China there are 7 species and 2 variants, of which only he first Wu, Prithia He shou wu, hairve stoic and toothleaf yorium belong to perennial herb and have a large root or root stem.
their appearance characteristics and physical and chemical characteristics are similar, the traditional identification methods through the eye, taste, nose, etc. are subjective, difficult to identify.
study selected 59 samples and 18 samples from 13 provinces in Anhui, Zhejiang, Henan, Guangxi, Guangdong, Sichuan, Guizhou, Hunan, Hubei, Yunnan, Jiangxi, Shaanxi and Gansu provinces.
Zheng Chuanjin and so on, based on the trnL-trnF sequence using PCR-RFLP technology can distinguish between He Shouwu and mixed counterfeits, but the whole process is long and expensive.
and alleles of gene-specific PCR identification of Chinese herbal medicine has simple and practical advantages, can effectively identify the authenticity of Ho Wu.
psbA-trnH DNA sequence has the characteristics of highly conservative within the species and sufficient interspecies variation .
this study by analyzing the difference sepsis DNA sequences between he And he's First Wu psbA-trnH, and tried to design and identify the primers of Ho's first wu, but it was very difficult because the two sequences were very similar.
after continuous attempts, a specific product is successfully designed, i.e. the method of introducing a mismatched base at the 3' end to design a specific primer.
at the same time, the product of AS-PCR amplification can be identified directly with agar gel and does not require sequencing.
this method is simple, convenient and short in time.
there are many factors that affect allele-specific PCR, the most important of which is annealing temperature, primer concentration and DNA template quantity, and a poor ratio of primer and primer to annealing temperature will cause nonspecific amplification, with false positives.
therefore, this study not only carried out the optimization experiment and systematic investigation of various factors on He Shouwu's specific PCR identification method, and determined the best PCR amplification reaction system, and all the herbs in this experiment were strictly morphologically identified, and the sample scores of He Shouwu genuine and counterfeit.