Identification of Dendrobium Species by Dot Blot Hybridization Assay

Abstract

The genus Dendrobium is one of the largest groups of the family Orchidaceae, which contains more than 1500 species distributed in different parts of the world. In China, the genus Dendrobium is composed of 74 species and two varieties, 1) of which the fresh or dried stem of D. nobile, D. officinale, D. fimbiratum and similar species are listed in the Chinese pharmacopoeia as Caulis Dendrobii (Shihu).2) Further, the dried Shihu is divided into two classes -"Huangcao Shihu" and "Fengdou Shihu" according to different botanical source and processing methods. The fresh stems without leaves or flowers of Dendrobium species, especially the processed and dried stems of commercial Shihu, which often come from a variety of Dendrobium species mixed together, have very similar morphological and anatomical characteristics, thus the authentication of different Dendrobium species using the traditional methods is very difficult.Plant species and Chinese medicinal materials identification methods currently used include analysis of morphological, chemical and genetic data. In the last decades, molecular methods, especially DNA sequencing, have been increasingly used for the identification.3-6) High polymorphisms of rDNA can be easily found between inter-species and even intraspecies. The DNA sequences of several traditional Chinese medicines (TCMs) have been reported; identifying markers were generated via restriction fragment length polymorphism (RFLP) [7][8][9] or polymerase chain reaction (PCR) methods [10][11][12] based on the polymorphic sequences. A system of designing PCR primers or probes is necessary to differentiate the TCMs in a quality control schedule. Such designed primers or probes would more easily produce identifying markers. Contaminated, broken and small amounts of DNA existing in TCMs frequently result in low reproducibility when molecular techniques are used. Therefore, highly sensitive and specific primers or probes are necessary to produce reliable results. Dot blot hybridization has been employed for the identification of microbes. Carlotti et al. described a rapid, reliable, and efficient tool for identifying 112 Candida krusei strains by dot blot hybridization using a species-specific probe-CkF1,2.13) The probe contained several middle repetitive sequences dispersed in the nontranscribed intergenic region of rDNA estimated to be approximately 10.6 kb in length. They used amounts as small as 60 to 120 ng of target DNA, and the species could be clearly detected by the probe with a nonradioactive label.13) Zeng et al. designed a series of species-specific ITS1-(internal transcribed spacer 1) and ITS2-based probes to successfully identify most of the 159 isolates of fungal pathogens from culture and clinical specimens in a reverse line blot hybridization assay.14) To rapidly authenticate species of TCMs, we need a simple method to deal with the large sample size on the market. We previously succeeded in authenticating different medicinal Dendrobium species by sequencing the rDNA ITS regions.15-18) However, t...

Effects of intracerebral hemorrhage on 5-hydroxymethylcytosine modification in mouse brains

“…DNA dot-blot assay was performed as described previously with some modifications. 25 Briefly, genomic DNA was spotted on a nitrocellulose membrane (Whatman plc, Maidstone, UK). The membrane was placed under an ultraviolet lamp for 20 minutes to crosslink the DNA and then blocked with 5% milk in Tris-buffered saline–Tween 20 for 1 hour, followed by incubation with the anti-5hmC (Active Motif, Carlsbad, CA, USA) antibody at 4°C overnight.…”

Deep Sequencing of Plant and Animal DNA Contained within Traditional Chinese Medicines Reveals Legality Issues and Health Safety Concerns

“…Likewise, genetic identification of animal species commonly involves DNA sequencing and characterisation of mitochondrial DNA (mtDNA) genes [1], [32], [34]. Despite the variety of genetic work that has been conducted to date, investigative research seems to have focused on detecting the DNA of specific targets within TCMs [22], [28], [30], [35]–[38] or herbal teas [39] rather than investigating all of the contributing species within a sample simultaneously.…”

Identification of Dendrobium Species Used for Herbal Medicines Based on Ribosomal DNA Internal Transcribed Spacer Sequence

“…[16][17][18][19][20][21][22][23][24][25][26][27] Unfortunately, these methods have only been applied to a limited number of Dendrobium species, and molecular markers that can be applied more universally to Dendrobium have not been developed. To recognize herbal medicines accurately at the species level, a comprehensive DNA sequence database that covers Dendrobium species potentially used for medicine is desired, and to create such a database, the following conditions are required: (1) precise identification of the plant species, (2) comprehensive data banking of reference sequences for the relevant plant group at the species level, and (3) employment of sequence markers that can discriminate interspecific differences.…”