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Convenient, sensitive and high-throughput method for screening botanic origin.

ABSTRACT: In this work, a rapid (within 4-5?h), sensitive and visible new method for assessing botanic origin is developed by combining loop-mediated isothermal amplification with cationic conjugated polymers. The two Chinese medicinal materials (Jin-Yin-Hua and Shan-Yin-Hua) with similar morphology and chemical composition were clearly distinguished by gene SNP genotyping assays. The identification of plant species in Patented Chinese drugs containing Lonicera buds is successfully performed using this detection system. The method is also robust enough to be used in high-throughput screening. This new method is very helpful to identify herbal materials, and is beneficial for detecting safety and quality of botanic products.

SUBMITTER: Yuan Y

PROVIDER: S-EPMC4066250 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Convenient, sensitive and high-throughput method for screening botanic origin.

Yuan Yuan Y Jiang Chao C Liu Libing L Yu Shulin S Cui Zhanhu Z Chen Min M Lin Shufang S Wang Shu S Huang Luqi L

Scientific reports 20140623

In this work, a rapid (within 4-5 h), sensitive and visible new method for assessing botanic origin is developed by combining loop-mediated isothermal amplification with cationic conjugated polymers. The two Chinese medicinal materials (Jin-Yin-Hua and Shan-Yin-Hua) with similar morphology and chemical composition were clearly distinguished by gene SNP genotyping assays. The identification of plant species in Patented Chinese drugs containing Lonicera buds is successfully performed using this de ...[more]

PMID: 24953704

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Project description:BackgroundMitochondrial dysfunction is linked to numerous pathological states, in particular related to metabolism, brain health and ageing. Nuclear encoded gene polymorphisms implicated in mitochondrial functions can be analyzed in the context of classical genome wide association studies. By contrast, mitochondrial DNA (mtDNA) variants are more challenging to identify and analyze for several reasons. First, contrary to the diploid nuclear genome, each cell carries several hundred copies of the circular mitochondrial genome. Mutations can therefore be present in only a subset of the mtDNA molecules, resulting in a heterogeneous pool of mtDNA, a situation referred to as heteroplasmy. Consequently, detection and quantification of variants requires extremely accurate tools, especially when this proportion is small. Additionally, the mitochondrial genome has pseudogenized into numerous copies within the nuclear genome over the course of evolution. These nuclear pseudogenes, named NUMTs, must be distinguished from genuine mtDNA sequences and excluded from the analysis.ResultsHere we describe a novel method, named MitoRS, in which the entire mitochondrial genome is amplified in a single reaction using rolling circle amplification. This approach is easier to setup and of higher throughput when compared to classical PCR amplification. Sequencing libraries are generated at high throughput exploiting a tagmentation-based method. Fine-tuned parameters are finally applied in the analysis to allow detection of variants even of low frequency heteroplasmy. The method was thoroughly benchmarked in a set of experiments designed to demonstrate its robustness, accuracy and sensitivity. The MitoRS method requires 5 ng total DNA as starting material. More than 96 samples can be processed in less than a day of laboratory work and sequenced in a single lane of an Illumina HiSeq flow cell. The lower limit for accurate quantification of single nucleotide variants has been measured at 1% frequency.ConclusionsThe MitoRS method enables the robust, accurate, and sensitive analysis of a large number of samples. Because it is cost effective and simple to setup, we anticipate this method will promote the analysis of mtDNA variants in large cohorts, and may help assessing the impact of mtDNA heteroplasmy on metabolic health, brain function, cancer progression, or ageing.

Project description:Targeted mass spectrometry (MS) approaches, which are powerful methods for uniquely and confidently quantifying a specific panel of proteins in complex biological samples, play a crucial role in validation and clinical translation of protein biomarkers discovered by global proteomic profiling. Common targeted MS methods, such as multiple reaction monitoring (MRM) and parallel-reaction monitoring (PRM), employ specific mass spectrometric technologies to quantify protein levels by comparing the transitions of specific endogenous (Endo) peptides with those of stable isotope labeled (SIL) peptide counterparts. These methods utilizing amino acid analyzed (AAA) SIL peptides warrants sensitive and precise measurements required for targeted MS assays. Compared to MRM, PRM provides higher experimental throughput by simultaneously acquiring all transitions of the target peptides and thereby compensate for different ion suppression among transitions of a target peptide. However, PRM still suffers different ion suppressions between Endo and SIL peptides due to poor spray stability as the Endo and SIL peptides were monitored at different liquid chromatography (LC) retention times. Here we introduce a new targeted MS method, termed as wideband PRM (WBPRM), designed for high-throughput targeted MS approach. WBPRM employs a wide isolation window for simultaneous fragmentations of both Endo and SIL peptides along with multiplexed single ion monitoring (SIM) scans for enhanced MS sensitivity of target peptides. Compared to PRM, WBPRM was demonstrated to provide increased sensitivity, precision, and accuracy of quantitative measurements of target peptides with increased throughput, allowing more target peptide measurements in a short experiment time. Its adaptability to a MS method provided by the manufacture makes it a facile method to implement for MS based assays, particularly in complex biological samples, where the needs for higher accuracy, sensitivity and efficiency are paramount.

Dataset Information

Convenient, sensitive and high-throughput method for screening botanic origin.

Publications

Convenient, sensitive and high-throughput method for screening botanic origin.

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