scholarly journals Verification of Thai ethnobotanical medicine “Kamlang Suea Khrong” driven by multiplex PCR and powerful TLC techniques

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257243
Author(s):  
Suthira Yanaso ◽  
Ampai Phrutivorapongkul ◽  
Darunee Hongwiset ◽  
Sirivipa Piyamongkol ◽  
Aekkhaluck Intharuksa
Keyword(s):  
Chemical Analysis ◽  
Multiplex Pcr ◽  
Plant Species ◽  
Specific Primers ◽  
Discrimination Power ◽  
Plant Materials ◽  
Identification Methods ◽  
Crude Drug ◽  
Crude Drugs ◽  
Species Specific

Kamlang Suea Khrong (KSK) crude drug, a traditional Thai medicine used for oral tonic and analgesic purposes, is obtained from three origins: the inner stem bark of Betula alnoides (BA) or the stems of Strychnos axillaris (SA) or Ziziphus attopensis (ZA). According to the previous reports, SA contains strychnine-type alkaloids that probably cause poisoning; however, only organoleptic approaches are insufficient to differentiate SA from the other plant materials. To ensure the botanical origin of KSK crude drug, powerful and reliable tools are desperately needed. Therefore, molecular and chemical identification methods, DNA barcoding and thin-layer chromatography (TLC), were investigated. Reference databases, i.e., the ITS region and phytochemical profile of the authentic plant species, were conducted. In case of molecular analysis, multiplex polymerase chain reaction (PCR) based on species-specific primers was applied. Regarding species-specific primers designation, the suitability of three candidate barcode regions (ITS, ITS1, and ITS2) was evaluated by genetic distance using K2P model. ITS2 presented the highest interspecific variability was verified its discrimination power by tree topology. Accordingly, ITS2 was used to create primers that successfully specified plant species of authentic samples. For chemical analysis, TLC with toluene:ethyl acetate:ammonia (1:9:0.025) and hierarchical clustering were operated to identify the authentic crude drugs. The developed multiplex PCR and TLC methods were then applied to identify five commercial KSK crude drugs (CK1-CK5). Both methods correspondingly indicated that CK1-CK2 and CK3-CK5 were originated from BA and ZA, respectively. Molecular and chemical approaches are convenient and effective identification methods that can be performed for the routine quality-control of the KSK crude drugs for consumer reliance. According to chemical analysis, the results indicated BA, SA, and ZA have distinct chemical profiles, leading to differences in pharmacological activities. Consequently, further scientific investigations are required to ensure the quality and safety of Thai ethnobotanical medicine known as KSK.

scholarly journals Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution

2004 ◽  
Vol 70 (5) ◽  
pp. 3171-3175 ◽  
Author(s):  
X. Bonjoch ◽  
E. Ballesté ◽  
A. R. Blanch
Keyword(s):  
16S Rrna ◽  
Multiplex Pcr ◽  
Fecal Pollution ◽  
Sensitivity Threshold ◽  
Rrna Gene ◽  
Specific Primers ◽  
Animal Wastewater ◽  
Wastewater Samples ◽  
Species Specific ◽  
Different Origins

ABSTRACT Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

Development of a multiplex PCR assay for the detection of key genes associated with Pasteurella multocida subspecies

2021 ◽  
pp. 104063872110634
Author(s):  
Barbara Ujvári ◽  
Hubert Gantelet ◽  
Tibor Magyar
Keyword(s):  
Multiplex Pcr ◽  
Pasteurella Multocida ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Multiplex Pcr Assay ◽  
Subspecies Level ◽  
Species Specific ◽  
Reference Strains

The ability to distinguish among the subspecies of Pasteurella multocida isolates is important epidemiologically; however, classification at the subspecies level based on the results of conventional biochemical tests (fermentation of sorbitol and dulcitol) is reportedly not accurate in all cases. Therefore, we developed a rapid, multiplex PCR assay to differentiate among the 3 subspecies of P. multocida. The PCR assay includes the P. multocida species–specific primers KMT1SP6 and KMT1T7 as an internal amplification control, with a newly designed gatD (galactitol-1-phosphate-5-dehydrogenase)-specific primer pair (unique for subsp. gallicida), and primers targeting a 16S rRNA gene region specific for subsp. septica. The subspecies specificity of the PCR was demonstrated by applying the test to a collection of 70 P. multocida isolates, including the Heddleston serovar reference strains; all isolates and strains were assigned correctly. The PCR assay is a sensitive, specific, and highly effective method for the identification of P. multocida subspecies, and an alternative to biochemical test–based differentiation. A possible relationship was noticed between P. multocida subspecies and lipopolysaccharide (LPS) genotype; all but one of the subsp. gallicida strains were isolated only from avian hosts and represented L1 LPS genotype. Subsp. multocida and subsp. septica isolates were classified into 5 and 4 different LPS genotypes, respectively, of which L3 was the only LPS genotype shared between these 2 subspecies.

scholarly journals Authentication of Shan-Dou-Gen Based on DNA Analysis

2019 ◽  
Vol 14 (6) ◽  
pp. 1934578X1985854
Author(s):  
Masashi Kitamura ◽  
Naoki Muramatsu ◽  
Ryuichiro Suzuki ◽  
Yoshiaki Shirataki
Keyword(s):  
Dna Analysis ◽  
Genetic Approach ◽  
Chain Reaction ◽  
Analgesic Effects ◽  
Specific Polymerase Chain Reaction ◽  
Crude Drug ◽  
Crude Drugs ◽  
Sophora Tonkinensis ◽  
Polymerase Chain ◽  
Species Specific

Shan-Dou-Gen (山豆根) is a crude drug mainly derived from the roots of Leguminosae plants, and it has antipyretic, antidotal, anti-inflammatory, and analgesic effects. In Japan, the root of Euchresta japonica has been used as a material of Shan-Dou-Gen. However, E. japonica is not used for medicinal purposes today, and commercial Shan-Dou-Gen products are imported from China. In China, several plant species have been used as Shan-Dou-Gen materials, but a crude drug derived from the root of Sophora tonkinensis is now officially used as Shan-Dou-Gen. However, it is difficult to morphologically identify the species used in Shan-Dou-Gen. In the present study, we showed that the Shan-Dou-Gen products commercially available in Japan are derived from S. tonkinensis using phylogenetic and sequencing analyses of the chloroplast trnH–psbA region. Furthermore, we performed species-specific polymerase chain reaction using conserved sequences of S. tonkinensis. Amplification was observed with Shan-Dou-Gen, whereas no amplification occurred with other crude drugs derived from the roots of S. flavescens and S. japonica. These results indicated that the genetic approach can be useful to authenticate Shan-Dou-Gen.

scholarly journals Molecular Detection of Phytophthora ramorum, the Causal Agent of Sudden Oak Death in California, and Two Additional Species Commonly Recovered from Diseased Plant Material

2004 ◽  
Vol 94 (6) ◽  
pp. 621-631 ◽  
Author(s):  
Frank N. Martin ◽  
Paul W. Tooley ◽  
Cheryl Blomquist
Keyword(s):  
Plant Species ◽  
The Other ◽  
Sudden Oak Death ◽  
Specific Primer ◽  
Specific Primers ◽  
Marker System ◽  
Phytophthora Spp ◽  
Multiplex Amplification ◽  
Species Specific ◽  
Specific Amplicon

Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. The pathogen causing this is Phy-tophthora ramorum, although species such as P. nemorosa and P. pseudo-syringae often are recovered from symptomatic plants as well. A molecular marker system was developed based on mitochondrial sequences of the cox I and II genes for detection of Phytophthora spp. in general, and P. ramorum, P. nemorosa, and P. pseudosyringae in particular. The first-round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for polymerase chain reaction (PCR) amplification and the other specific for amplification of sequences from Phytophthora spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. Using DNA from purified cultures, the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species. The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species. With the exception of one plant species, the resulting amplicons were smaller than the Phytophthora genus-specific amplicon. The products of the first-round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P. ramorum, P. nemorosa, or P. pseudo-syringae. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P. ramorum, this included 24 isolates from California, Germany, and the Netherlands. Using purified pathogen DNA, the limit of detection for P. ramorum using this marker system was ≈2.0 fg of total DNA. However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure, the sensitivity of detection was reduced by 100- to 1,000-fold, depending on the plant species. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions and, for P. ramorum, amplification with a previously published rDNA internal transcribed spacer species-specific primer pair. Results were compared and validated with three different brands of thermal cyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions. The specificity of the Phytophthora genus-specific primers suggests that they will have utility for pathogen detection in other Phytophthora pathosystems.

scholarly journals Biotyping and Antimicrobial Susceptibility of Enterococcus faecalis and E. faecium Isolated from Urine and Stool Samples

2021 ◽  
Vol 13 (10) ◽  
Author(s):  
Gülşah Tollu ◽  
Ismail Hakkı Ekin
Keyword(s):  
Multiplex Pcr ◽  
Antimicrobial Susceptibility ◽  
Animal Health ◽  
Molecular Techniques ◽  
Penicillin G ◽  
Clinical Approach ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Stool Samples ◽  
Species Specific

Background: Enterococci are one of the opportunistic pathogenic microorganisms that can cause significant problems for human and animal health. Enterococcus faecium seems to be more resistant to antibiotics than E. faecalis. It is thought that pathogenic E. faecium can develop antibiotic resistance very quickly, and the ability to transfer this feature is considered to be an important health risk. Objectives: This study aimed to determine the prevalence, biotypes, and in vitro antimicrobial susceptibility of E. faecalis and E. faecium strains isolated from 267 routine urine and stool samples that were brought to the microbiology laboratory of Regional Training and Research Hospital of Van, with permission of the patients. Methods: In the present study, enterococci using species-specific primers to examine E. faecalis and E. faecium multiplex PCR technique was applied. Biotyping of the isolates was used to identify them as E. faecalis and E. faecium by molecular techniques, and antibiotic susceptibility of all samples was examined, as well. Results: The isolates were identified by multiplex PCR using species-specific primers for E. faecalis and E. faecium. Biotyping based on 13 biochemical tests showed that 72.5%, 12.5%, and 15% of E. faecalis strains were of biotypes I, II, and III, respectively, whereas E. faecium strains could be divided into biotype I (10%), biotype II (12.5%), biotype III (27.5%), and biotype IV (50%). Additionally, all E. faecalis strains were found to be susceptible to penicillin G and imipenem. On the other hand, 95% of the E. faecalis strains were found to be resistant to clindamycin, 77.5% to tetracycline and trimethoprim/sulfamethoxazole, 42.5% to erythromycin, 32.5% to gentamicin, and 17.5% to ciprofloxacin. Of E. faecium strains, 37.5% were found to be resistant to clindamycin, 32.5% to penicillin G, 27.5% to erythromycin and imipenem, 20% to ciprofloxacin, 17.5% to tetracycline and trimethoprim/sulfamethoxazole, 15% to gentamicin, and 5% to vancomycin. Conclusions: In conclusion, the identification of E. faecalis and E. faecium strains by PCR is reliable and faster than biochemical tests. Additionally, the results of antimicrobial susceptibility tests may provide important contributions to the clinical approach.

Agronomically important thrips: development of species-specific primers in multiplex PCR and microarray assay using internal transcribed spacer 1 (ITS1) sequences for identification

2014 ◽  
Vol 105 (1) ◽  
pp. 52-59 ◽  
Author(s):  
W.B. Yeh ◽  
M.J. Tseng ◽  
N.T. Chang ◽  
S.Y. Wu ◽  
Y.S. Tsai
Keyword(s):  
Multiplex Pcr ◽  
Species Identification ◽  
Internal Transcribed Spacer ◽  
High Throughput Screening ◽  
Virus Transmission ◽  
Specific Primers ◽  
Its1 Sequence ◽  
Microarray Assay ◽  
Thrips Species ◽  
Species Specific

AbstractThrips, the sole vector of plantTospovirus, are major pests of many agricultural crops throughout the world. Molecular approaches have been applied in recent decades to identify these minute and morphologically difficult to distinguish insects. In this study, sequences of internal transcribed spacer 1 (ITS1) region of 15 agronomically important thrips, including several virus transmission species, have been analyzed in order to design species-specific primers for multiplex PCR and probes for microarray assay. That the ITS1 sequence distances within species were smaller than those among species suggests that the ITS1 fragment can be used for thrips species identification. The specificity and stability of these primers, combined with universal paired primers, were tested and verified in multiplex PCR. Using these specific primers as probes, microarray assay showed that PCR products of all thrips species hybridized consistently to their corresponding probes, though some signals were weak. We have demonstrated that multiplex PCR using specific primers based on ITS1 sequences is a simple, reliable, and cost-effective diagnostic tool for thrips species identification. Moreover, the DNA microarray assay is expected to extend into a reliable high-throughput screening tool for the vast numbers of thrips.

Rapid identification of Saccharomonospora strains by multiplex PCR using species-specific primers within the 16S rRNA gene

1996 ◽  
Vol 27 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Jung-Hoon Yoon ◽  
Sung Taik Lee ◽  
Yong Kook Shin ◽  
Sam-Bong Kim ◽  
Hong-Joong Kim ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Multiplex Pcr ◽  
Rapid Identification ◽  
Rrna Gene ◽  
Specific Primers ◽  
Species Specific ◽  
The 16S Rrna Gene

scholarly journals A Multiplex PCR for the Detection of Phytophthora nicotianae and P. cactorum, and a Survey of Their Occurrence in Strawberry Production Areas of Japan

Plant Disease ◽  
2011 ◽  
Vol 95 (10) ◽  
pp. 1270-1278 ◽  
Author(s):  
Mingzhu Li ◽  
Takahiro Asano ◽  
Haruhisa Suga ◽  
Koji Kageyama
Keyword(s):  
Multiplex Pcr ◽  
Protein Gene ◽  
Phytophthora Nicotianae ◽  
Soilborne Pathogens ◽  
Chain Reaction ◽  
Specific Primers ◽  
Phytophthora Spp ◽  
Polymerase Chain ◽  
Species Specific ◽  
Production Areas

We aimed to simultaneously detect two pathogens causing strawberry diseases, Phytophthora nicotianae and P. cactorum, by multiplex polymerase chain reaction (PCR), and to survey their occurrence in the main strawberry production areas of Japan. Due to the need to combine different primer pairs for multiplex PCR and the low specificity of published specific primers for P. nicotianae and P. cactorum, new species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions of ribosomal DNA and the ras-related protein gene Ypt1, respectively. Specificity of the designed primers was demonstrated using 68 isolates, including Phytophthora spp., Pythium spp., and other soilborne pathogens. Multiplex PCR discriminated between P. nicotianae and P. cactorum in DNA mixtures of mycelia of the two species. Moreover, both species were detected in artificially and naturally infested soils, indicating that these markers can be used in diagnosis of strawberry diseases. For investigation of the geographic distribution of the two pathogens in Japan, soil samples were collected in 89 strawberry fields from eight prefectures (Gifu, Saga, Nara, Tochigi, Chiba, Shizuoka, Yamanashi, and Hokkaido) of Japan. The method that was developed was successfully applied to survey P. nicotianae and P. cactorum, and distribution of the two pathogens in strawberry plantings in Japan was determined.

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