[Site-specific PCR identification of animal species and formula particles of Scorpio].

Scorpio, a commonly used animal medicine in China, is derived from Buthus martensii as recorded in the Chinese Pharmacopoeia. China harbors rich species of Scorpionida and adulterants exist in the raw medicinal material and deep-processed products of Scorpio. The microscopic characteristics of the deep-processed products may be incomplete or lost during processing, which makes the identification difficult. In this study, the maximum likelihood(ML) tree was constructed based on the morphology and cytochrome C oxidase subunit I(COⅠ) to identify the species of Scorpio products. The results showed that the main adulterant of Scorpio was Lychas mucronatus. According to the specific SNP sites in the COⅠ sequence of B. martensii, the stable primers were designed for the identification of the medicinal material and formula granules of Scorpio. The polymerase chain reaction(PCR) at the annealing temperature of 61 ℃ and 30 cycles produced bright specific bands at about 150 bp for both B. martensii and its formula particles and no band for adulterants. The adaptability of the method was investigated, which showed that the bands at about 150 bp were produced for Scorpio medicinal material, lyophilized powder, and formula granules, and commercially available formula granules. The results showed that the established method could be used to identify the adulterants of Scorpio and its formula granules, which could help to improve the quality control system and ensure the safe clinical application of Scorpio formula granules.

[Identification of Cervi Cornu (Cervus elaphus) and its formula granules based on PCR-RFLP].

Cervi Cornu is the ossified antler, or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C. nippon, as a precious traditional Chinese medicine, has been recognized for its medicinal value and widely used in clinical practice. However, the origins of Cervi Cornu are miscellaneous, and Cervi Cornu is even mixed with adulterants in the market. Currently, there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cornu. So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants. In this study, the differences in the mitochondrial barcode cytochrome b(Cytb) gene sequences of C. elaphus, C. nippon and their related species were compared and the specific single nucleotide polymorphism(SNP) sites on the Cytb sequences of Cervi Cornu were screened out. According to the screened SNPs, Cervi Cornu-specific primers dishmy-F and dishmy-R were designed. The PCR system was established and optimized, and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated. The amplification products of C. elaphus and C. nippon were digested using the restriction enzyme MseⅠ. The results showed that after electrophoresis of the product from PCR with the annealing temperature of 56 ℃ and 35 cycles, a single specific band at about 100 bp was observed for C. elaphus samples, and the product of C. elaphus samples was 60 bp shorter than that of C. nippon samples. There was no band for adulterants from other similar species such as Alces alces, Rangifer tarandus, Odocoileus virginianus, O. hemionus, Cap-reolus pygargus, Przewalskium albirostis and negative controls. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method established in this study can quickly and accurately identify Cervi Cornu originated from C. elaphus in crude drugs, standard decoctions, and formula granules, and distinguish the origins of Cervi Cornu products, i.e., C. nippon and similar species. This study can be a reference for other studies on the quality standard of other formula granules of traditional Chinese medicines.

Biosensing with Polymerase Chain Reaction-Stable DNA-Functionalized Magnetically Susceptible Carbon-Iron Microparticles.

We demonstrate a rapid and sensitive method for DNA detection without the need for fluorescence. This is based on carbon-coated magnetic iron (Fe) microparticles with a covalent surface attachment of DNA. We show that these magnetic microparticles can capture complementary DNA. Significantly, the DNA covalent surface bonds are robust to high temperatures and can be included in a sample during polymerase chain reaction (PCR). This method is employed for the detection of targeted DNA sequences (40-50 bp). Hybridization probes on the surface of the magnetically susceptible Fe microparticle recognize the target DNA sequence-specifically. The double-stranded DNA (dsDNA) microparticles are then quickly captured with a magnet from the sample matrix. This foregoes postpurification processes, such as electrophoresis, which make our technique time- and cost-effective. Captured dsDNA can be detected with intercalating dyes such as ethidium bromide through a loss in the UV absorption signal with a limit of detection (LOD) of 24 nM within 15 min. Likewise, surface-bound DNA can act as a primer in PCR to decrease the LOD to 5 pM within 2 h. This is the first instance of a nucleotide-modified magnetically susceptible carbon substrate that is PCR-compatible. Besides DNA capture, this strategy can eventually be applied to sequence-specific nucleic acid purification and enrichment, PCR cleanup, and single-strand generation. The DNA-coated particles are stable under PCR conditions (unlike commonly used polystyrene or gold particles).

ABSP: an automated R tool to efficiently analyze region-specific CpG methylation from bisulfite sequencing PCR.

MOTIVATION: Nowadays, epigenetic gene regulations are studied in each part of the biology, from embryonic development to diseases such as cancers and neurodegenerative disorders. Currently, to quantify and compare CpG methylation levels of a specific region of interest, the most accessible technique is the bisulfite sequencing PCR (BSP). However, no existing user-friendly tool is able to analyze data from all approaches of BSP. Therefore, the most convenient way to process results from the direct sequencing of PCR products (direct-BSP) is to manually analyze the chromatogram traces, which is a repetitive and prone to error task. RESULTS: Here, we implement a new R-based tool, called ABSP for analysis of bisulfite sequencing PCR, providing a complete analytic process of both direct-BSP and cloning-BSP data. It uses the raw sequencing trace files (.ab1) as input to compute and compare CpG methylation percentages. It is fully automated and includes a user-friendly interface as a built-in R shiny app, quality control steps and generates publication-ready graphics. AVAILABILITY AND IMPLEMENTATION: The ABSP tool and associated data are available on GitHub at https://github.com/ABSP-methylation-tool/ABSP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Nucleic acid amplification test (NAAT) conducted in a microfluidic chip to differentiate between various ginseng species.

Panax ginseng and Panax quinquefolius have different medicinal properties and market values; however, they can be difficult to distinguish from one another based on physical appearances alone. Therefore, a molecular test that can be performed in commercial settings is needed to overcome this difficulty. A locus that contains a single nucleotide polymorphism (SNP) site to differentiate between P. ginseng and P. quinquefolius has been selected. An isothermal nucleic acid amplification test (NAAT) has been developed for use in a microfluidic chip; this NAAT method, which is based on lesion-induced DNA amplification (LIDA), amplifies the extracted plant genomic samples and enhances the detection of specific SNPs. This NAAT method was used to authenticate five ginseng root samples which indicated that two of the five samples appear to be mislabeled. These authentication results were consistent with those obtained from next generation sequencing (NGS) although this molecular test is more affordable and faster than NGS.

A Lab-on-a-Chip Approach for the Detection of the Quarantine Potato Cyst Nematode Globodera pallida.

The potato cyst nematode (PCN), Globodera pallida, has acquired significant importance throughout Europe due to its widespread prevalence and negative effects on potato production. Thus, rapid and reliable diagnosis of PCN is critical during surveillance programs and for the implementation of control measures. The development of innovative technologies to overcome the limitations of current methodologies in achieving early detection is needed. Lab-on-a-chip devices can swiftly and accurately detect the presence of certain nucleotide sequences with high sensitivity and convert the presence of biological components into an understandable electrical signal by combining biosensors with microfluidics-based biochemical analysis. In this study, a specific DNA-probe sequence and PCR primers were designed to be used in a magnetoresistive biosensing platform to amplify the internal transcribed spacer region of the ribosomal DNA of G. pallida. Magnetic nanoparticles were used as the labelling agents of asymmetric PCR product through biotin−streptavidin interaction. Upon target hybridization to sensor immobilized oligo probes, the fringe field created by the magnetic nanoparticles produces a variation in the sensor's electrical resistance. The detection signal corresponds to the concentration of target molecules present in the sample. The results demonstrate the suitability of the magnetic biosensor to detect PCR target product and the specificity of the probe, which consistently distinguishes G. pallida (DV/V > 1%) from other cyst nematodes (DV/V < 1%), even when DNA mixtures were tested at different concentrations. This shows the magnetic biosensor's potential as a bioanalytical device for field applications and border phytosanitary inspections.

Detection of Ilyonectria pseudodestructans from Potato by RNase H-Dependent PCR (rhPCR) and rh-Quantitative PCR (rhqPCR).

Ilyonectria pseudodestructans, a plant pathogen that is known to cause root rot on fruit trees such as grapevine and apple, has recently been reported to also cause tuber decay in potato. The increasing risk of this pathogen on various horticultural crops makes it essential to develop a rapid and accurate detection method. In this study, an RNase H-dependent PCR (rhPCR) protocol and a modified probe-based rh-quantitative PCR (rhqPCR) protocol for I. pseudodestructans detection were developed. Both the forward and reverse primers for rhPCR and rhqPCR carry an RNA nucleotide at the site where a single-nucleotide polymorphism between I. pseudodestructans and strains of other Ilyonectria spp. is located, and the rhqPCR also contains a fluorescent-labeled target-specific probe. The primers were designed based on the sequence of the histone H3 gene and could amplify a DNA fragment of 73 bp. In the specificity test, by alignment via the BLASTn tool, the RNA nucleotide bases on both the forward and the reverse primers were identical to the corresponding genomic site of 16 of 17 (94.1%) database-available I. pseudodestructans strains, and different from 43 of 44 (97.7%) database-available strains of other Ilyonectria spp. When the rhPCR and rhqPCR protocols were applied on 11 I. pseudodestructans strains and 46 other strains of different species of plant pathogens, all of the I. pseudodestructans strains generated positive reactions whereas all of the other strains were negative, which indicated an excellent specificity of the primers. In the sensitivity test, the lowest DNA template amount for a positive reaction using the rhPCR and rhqPCR methods was 2 pg for I. pseudodestructans genomic DNA. When testing the rhqPCR method on gBlock, the lowest number of molecules for a positive reaction was six. These results indicated a high sensitivity of the protocol for I. pseudodestructans detection. To our knowledge, this is the first report of a probe-based rhqPCR to be applied to plant disease diagnosis; in addition, this is also the first rapid molecular protocol to detect I. pseudodestructans. The new rhPCR and rhqPCR methods have a potential to be applied by plant disease diagnostic labs for their routine work.

Molecular Analysis With 16S rRNA PCR/Sanger Sequencing and Molecular Antibiogram Performed on DNA Extracted From Valve Improve Diagnosis and Targeted Therapy of Infective Endocarditis: A Prospective Study.

BACKGROUND: Molecular analysis (MA) on heart valve (HV) improves the microbiologic diagnosis of infectious endocarditis (IE). The main drawback of MA is the lack of antimicrobial susceptibility information. METHODS: We conducted a prospective cohort observational study of consecutive adult patients from April 2012 to May 2021 who underwent valve surgery at our hospital. The performance of MA, blood cultures (BC) and valve cultures (VC), and the diagnostic and therapeutic impact of MA were evaluated. Molecular antibiogram results were compared to culture-based antimicrobial susceptibility testing (AST). RESULTS: A total of 137 patients with definite IE and 52 patients with no IE were enrolled in the study. Among IE cases BC, VC, and MA were positive in 75 (55%), 30 (22%), and 120 (88%) of IE cases, respectively. Among 62 cases of BC-negative IE (BCNE), 57 achieved diagnosis with MA. MA led to a change of antimicrobial therapy in 92% of BCNE. MA was negative in 100% of patients with no IE. Molecular antibiogram performed on 17 valve specimens that resulted positive for pathogens potential carrier of genes encoding for multidrug resistant mechanisms showed 100% concordance with AST. CONCLUSIONS: MA showed a high specificity and sensitivity in etiological diagnosis of IE. Molecular antibiogram could overcome the major limitation of MA that is the lack of susceptibility testing. We advocate for the inclusion of MA among diagnostic criteria for IE and for a more extensive use of molecular antibiogram when the culture result is negative, and MA is the only positive test.

Comparative analysis of microsatellite instability by next-generation sequencing, MSI PCR and MMR immunohistochemistry in 1942 solid cancers.

Checkpoint inhibitor approval for microsatellite instability-high (MSI-H) tumours has made MSI as a therapeutically important biomarker. Next-generation sequencing (NGS)-based MSI detection is being widely used for assessing MSI. However, MSI tumours detected using NGS and their relevance to MSI-polymerase chain reaction (PCR) and mismatch repair deficiency (dMMR) are unclear. In 1942 solid cancer cases tested using NGS-based comprehensive cancer panel with 523 genes (1.94 mb), the MSI score, tumour mutation burden (TMB; ≥ 10 mutations/mb), and frameshift mutations were analysed. GeneScan analyses of five mononucleotide markers (MSI-PCR) and MMR protein immunohistochemistry (IHC) were compared with the NGS-MSI results. With a ≥ 12% MSI score as a cut-off for MSI-H, two MSS cases were classified as MSI-H. With a ≥ 20% cut-off, 10 cases categorised as MSS by NGS were MSI-H/dMMR by MSI-PCR and MMR IHC. To avoid discrepant cases, we adopted a high MSI cut-off and a borderline MSI category. Finally, MSI-H (≥ 20%), borderline MSI (≥ 7% and < 20%), and MSS (< 7%) were found in 35 (1.8%), 24 (1.2%), and 1883 (97%) cases, respectively. All MSI-H cases by NGS were MSI-H/dMMR by MSI-PCR and MMR IHC. Of the 24 borderline MSI cases by NGS, MSI-H/dMMR was 9 (37.5%) cases, MSS/dMMR was 1 (4.2%) case, and 11 (45.8%) of them had high TMB. All MSS cases by NGS were MSS/pMMR by MSI-PCR/IHC, and 257 (13.6%) had high TMB. With those arbitrary cut-off points, 10 (0.5%) MSS cases using NGS were discrepant with MSI-PCR or MMR IHC, and all were borderline MSI cases. The mean number of frameshift mutations was significantly higher in the MSI-H group (28.3) than in the borderline MSI (7.7) or MSS (1.3) groups (p < 0.001). In conclusion, to facilitate therapeutic decision-making for NGS, cut-off points for MSI can be defined based on MSI-PCR/dMMR confirmation.

One-Enzyme RTX-PCR for the Detection of RNA Viruses from Multiple Virus Genera and Crop Plants.

Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

Microdissection Methods Utilizing Single-Cell Subtype Analysis and the Impact on Precision Medicine.

Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology field where tumor genotyping is often essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Utilizing advanced stage lung cancer as an example, an improved genotyping approach for solid tumors is possible. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from formalin-fixed paraffin-embedded (FFPE) specimens sharing similar characteristics, in other words, single-cell subtype analysis. The shared characteristics can include immunostaining status, cell phenotype, and/or spatial location within a histological section. Synergy between microdissection and droplet digital PCR (ddPCR) enhances the molecular analysis. We demonstrate here a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time- and cost-efficient manner, using immunostain targeting as an example.

Development and Validation of a Species-specific PCR Method for the Identification of Ginseng Species Using Orthogonal Approaches.

When testing botanical ingredients of herbal medicines and dietary supplements, the complexity of botanical matrixes often requires the use of orthogonal methods to establish identification procedures suitable for quality control purposes. Genomic-based botanical identification methods are evolving and emerging as useful quality control tools to complement traditional morphological and chemical identification methods. Species-specific polymerase chain reaction methods are being evaluated for botanical quality control and as a cost-effective approach to identify and discriminate between closely related botanical species. This paper describes orthogonal identification of Panax ginseng, P. quinquefolius, and P. notoginseng materials in commerce as an example of the development and validation of a set of species-specific polymerase chain reaction methods to establish botanical identity in ginseng roots. This work also explored the possibility of extending the application of species-specific polymerase chain reaction methods to provide species identity information for processed materials, such as steamed roots and hydroalcoholic extracts, and showed success with this approach. Finally, the paper provides recommendations for an out-of-specification investigation of samples that may pass some of the orthogonal tests and fail others.

Peptide nucleic acid as a template for Taq DNA polymerase.

Peptide nucleic acid (PNA), an artificial DNA analog, comprises a purine or pyrimidine base and a pseudo-peptide backbone instead of deoxyribose-phosphate. PNA has been found to have stronger adhesion and higher stability in binding to its complementary DNA than deoxyribose-phosphate. Thus, it could serve as an agent for gene modulation, demonstrating potential in antisense therapy, molecular diagnostics, and nanotechnology. However, the applications of PNA remain limited because its biological activities are not fully known. Here, I demonstrate that a thermostable DNA polymerase, Thermus aquaticus (Taq) polymerase, exhibits transcriptase activity when a PNA oligomer is used as a template and that genetic information of the oligomer can be amplified by PCR using DNA primers. Furthermore, the insertion of a glutamine peptide stretch in the middle part of the PNA template did not interfere with transcription; it was transcribed into a guanosine or adenosine stretch. Intriguingly, this amino acid-to-DNA transcription did not occur when glycine residues were inserted. A synthetic PNA oligomer can, therefore, function as a template for a DNA polymerase, and polyglutamine peptides can be transcribed into guanosine or adenosine. These findings provide a cornerstone to reveal all amino acid genetic codes and transcription activity in the future.

High-throughput measuring of meiotic recombination rates in barley pollen nuclei using Crystal Digital PCRTM.

Breeding exploits novel allelic combinations assured by meiotic recombination. Barley (Hordeum vulgare) single pollen nucleus genotyping enables measurement of meiotic recombination rates in gametes before fertilization without the need for segregating populations. However, so far, established methods rely on whole-genome amplification of every single pollen nucleus due to their limited DNA content, thus restricting the number of analyzed samples. In this study, high-throughput measurements of meiotic recombination rates in barley pollen nuclei without whole-genome amplification were performed through a Crystal Digital PCRTM -based genotyping assay. Meiotic recombination rates within two centromeric and two distal chromosomal intervals were measured in hybrid plants by genotyping a total of >42 000 individual pollen nuclei (up to 4900 nuclei analyzed per plant). Determined recombination frequencies in pollen nuclei were similar to frequencies in segregating populations. We improved the efficiency of the genotyping by pretreating the pollen nuclei with a thermostable restriction enzyme. Additional opportunities for a higher sample throughput and a further increase of the genotyping efficiency are presented and discussed. Taken together, single barley pollen nucleus genotyping based on Crystal Digital PCRTM enables reliable, rapid and high-throughput meiotic recombination measurements within defined chromosomal intervals of intraspecific hybrid plants. The successful encapsulation of nuclei from a range of species with different nuclear and genome sizes suggests that the proposed method is broadly applicable to genotyping single nuclei.

ITS 2 and RNA secondary structure-based analysis reveals a clear picture on phylogeny of South Indian Salacia spp.

The genus Salacia (Celastraceae) consists of many important medicinal plants used mainly against type II diabetes. Segregation and delimitation of species is difficult based on morphological features alone. DNA barcoding is the most effective and emerging method of molecular identification. It was reported that ITS2 has better discriminating power in the genus Salacia in comparison to other barcode loci. This paper describe the analysis of sequence and structural information of ITS2 to discriminate the species of Salacia. A total of 8 species of Salacia in South India and the available sequences in NCBI database were taken for the present study. NJ method based phylogenetic trees were constructed using MEGAX with primary sequence as well as using sequence and secondary structural information. Primary structure based phylogeny did not give much information whereas the dendrogram based on sequence and structural information was more informative to decipher the phylogeny of South Asian species of Salacia. The present study revealed some interesting facts regarding the genus. Secondary structure of the ITS2 sequence of S. chinensis reported from Kerala differs consistently from that of S. chinensis reported from other parts of India and of South Asia. Probably the S. chinensis in Kerala, India has diverged a lot from the original S. chinensis. ITS2 sequence of S. reticulata reported from Sri Lanka was identical to S. chinensis reported by other groups from Thailand and Udupi, India. The molecular level identity of ITS2 sequence of S. chinensis with S. reticulata suggest merger of the two species. ITS2 sequence of S. beddomei is only reported from Kerala, India showed it to be identical to S. macrosperma. This observation points to a mistaken identity of S. beddomei which could be elusive from Kerala. Phylogenetic trees constructed based on sequence and structural features of ITS2 suggest that the ancestor species of S.chinensis diversified in two evolutionary lines. One line leads to the present day S. chinensis and the other line further diversified and lead to the rest of the present day Salacia species.

Quantification of Actaea racemosa L. (black cohosh) from some of its potential adulterants using qPCR and dPCR methods.

The demand for popular natural health products (NHPs) such as Black Cohosh is increasing considerably, which in turn challenges quality assurance (QA) throughout the supply chain. To detect and quantify the target species present in a given NHP, DNA-based molecular techniques such as Real-time quantitative PCR (qPCR) and digital PCR (dPCR) are standard tools in the food and pathogen testing industries. There is a gap in the literature concerning validated quantitative PCR methods for botanicals that can be utilized for QA and good manufacturing practices. The objective of this study is to develop an efficient quantification method using qPCR and dPCR techniques for the detection and quantification of Actaea racemosa (Black cohosh) NHPs from its potential adulterants. These developed methods are validated for applicability on commercial NHPs. Species-specific hydrolysis probe assays were designed to analyze the black cohosh NHPs using qPCR and dPCR techniques. The results confirmed that the developed qPCR and dPCR methods are highly precise for identifying and quantifying black cohosh NHPs, indicating their potential applicability in future routine industrial and laboratory testing. This enables a single qPCR test to determine not only the presence of a specific botanical, but also the amount when mixed with an adulterant.

Genetic identification of medicinally used Salacia species by nrDNA ITS sequences and a PCR-RFLP assay for authentication of Salacia-related health foods.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots and stems of several Salacia species have been used as traditional medicines, especially in Ayurvedic medical system for the treatment of diabetes, rheumatism, gonorrhea, amenorrhea, skin diseases, etc. Due to reported evidence supporting Salacia's beneficial effects in early-stage diabetes and other lifestyle-related diseases, Salacia-based dietary supplements and health foods have been gaining popularity in Japan and other countries in recent years. However, due to the morphological similarities between Salacia plants, particularly in the medicinally used parts (roots and stems), the authentication of the botanical identities of Salacia-derived products is challenging. AIM OF THIS STUDY: This study aims to develop a genetic approach to authenticate the medicinally used Salacia species and to determine the botanical sources of the commercially available Salacia-derived products. MATERIALS AND METHODS: The sequences of nuclear DNA internal transcribed spacer (ITS) and chloroplast trnK-rps16 region were determined and compared between 10 plant specimens from three medicinally used Salacia species as well as 48 samples of commercial crude drugs. Moreover, a PCR-restriction fragment length polymorphism (RFLP) assay was developed for rapid identification based on the ITS sequences. RESULTS: The plant specimens from the three medicinally used Salacia species showed three main types of sequences in both ITS (types I, II, III) and trnK-rps16 (i, ii, iii) regions. Combined the sequences of ITS and trnK-rps16 regions, S. reticulata and S. oblonga had type I-i and type III-iii or similar sequences, respectively. S. chinensis had type II-ii or II(536M)-i sequences. Forty-eight samples of commercial crude drugs were identified based on ITS and trnK-rps16 DNA barcode. A convenient PCR-RFLP assay using Cac8I restriction enzyme was established and applied to identify the botanical sources of health food products purchased from online retailers. All the twelve samples were identified as S. chinensis. CONCLUSION: The nrDNA ITS sequences provided useful information to authenticate Salacia species and to elucidate the phylogenetic relationship within the Salacia genus. Genetic identification results revealed that S. chinensis and S. reticulata are the major sources of commercially available Salacia-products. Based on the ITS sequences, a convenient PCR-RFLP assay was established for the identification of the medicinally used Salacia species as well as their derived health food products.

Development of a species-specific PCR assay for authentication of Agkistrodon acutus based on mitochondrial cytochrome b gene

BACKGROUND: Agkistrodon acutus, a traditional Chinese medicine, clinically used in the treatment of rheumatism, tumor, and cardiovascular and cerebrovascular diseases. Due to the unique medicinal value and the difficulty of artificial breeding of Agkistrodon acutus, the supply of Agkistrodon acutus on the market exceeds the demand, and a large number of its adulterants are found on the market. In this study, the cytb gene sequences of Agkistrodon acutus and 9 snakes were compared and analyzed, specific primers were designed, and specific PCR methods were established to detect Agkistrodon acutus medicinal samples on the market. RESULTS: This method was successfully applied to distinguish the snake from other adulterated species, and tested 18 Agkistrodon acutus samples randomly purchased from six cities. Twelve samples were counterfeit and six were genuine. The standard reference material of Agkistrodon acutus was cloned by molecular cloning and sequencing, and the gene sequence difference with other species was significant. It shows that the region could be used as the fingerprint region of the target species. CONCLUSIONS: The proposed method can be used as a species-specific marker and can be highly distinguished from other adulterated snake species, which is helpful to effectively avoid the problem of false sale of Agkistrodon acutus.

Hairpin-Bisulfite PCR.

Ligation of a hairpin oligonucleotide to genomic DNA prior to bisulfite conversion and PCR amplification physically links the two complementary DNA strands. This additional step in the conversion procedure overcomes the limitations of conventional bisulfite sequencing where information of the cytosine methylation status is only obtained from one of the two strands of an individual DNA molecule. Sequences derived from hairpin bisulfite PCR products reveal the dynamics of this epigenetic memory system on both strands of individual DNA molecules. The chapter describes a reliable step-by-step procedure to generate hairpin-linked DNA. It also provides a guide for efficient bisulfite conversion that is suitable for both conventional and hairpin bisulfite sequencing approaches.

Specific PCR-based detection of Phomopsis heveicola the cause of leaf blight of Coffee (Coffea arabica L.) in China.

Coffee (Coffea arabica L.) is currently grown in many tropical and subtropical areas countries and is a major traded commodity for the developing world. Coffee leaf blight, caused by Phomopsis heveicola, is one of the most important fungal diseases dangerous to coffee crops in China. This study aimed to develop a PCR-based diagnostic method for detecting P. heveicola in planta. Specific primers (CPHF/CPHR) were designed based on sequence data of region of internal transcribed spacer (ITS1 and ITS4) of P. heveicola. The efficiency and specificity of CPHF/CPHR were established by PCR analysis of DNA from P. heveicola strains isolated from China and fungal isolates of other genera. A single amplification product of 318 bp was detected from DNA P. heveicola isolates. No amplification product was observed with any of the other fungal isolates tested. The specific primers designed and employed in PCR detected P. heveicola up to 3 pg from DNA isolated. This is the first report on the development of a species-specific PCR assay for identification and detection of P. heveicola. Thus, the PCR-based assay developed was very specific, rapid and sensitive tool for the detection of pathogen P. heveicola.