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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.
Subject(s)
Animals , Polymerase Chain Reaction/methods , Agkistrodon/genetics , Cytochromes b/genetics , Mitochondria/genetics , Snakes , Species Specificity , DNA/analysis , Cloning, Molecular , Medicine, Chinese TraditionalABSTRACT
BACKGROUND In Acre state, Brazil, the dissemination of cutaneous leishmaniasis has increased in recent years, with limited knowledge of the potential Leishmania spp. vectors involved. OBJECTIVES Here, data concerning the sandfly fauna of Brasiléia municipality, Leishmania DNA-detection rates and the identification of blood meal sources of insects captured in 2013-2015 are presented. METHODS Parasite detection in female sandflies was performed individually by multiplex polymerase chain reaction (PCR) (Leishmania kDNA/sandfly cacophony-gene), with the identification of Leishmania spp. by hsp70-PCR and sequencing. The identification of blood gut-content from fed females was performed by cyt b-PCR and sequencing. FINDINGS A total of 4,473 sandflies were captured. A subgroup of 864 non-blood-fed females evaluated for the presence of Leishmania DNA showed 2.9% positivity for Leishmania (Viannia) braziliensis and L. (V.) guyanensis. The identification of blood meal sources was performed in 96 blood-fed females, allowing the identification of 13 vertebrate species. In nine/96 fed females, DNA from L. (V.) shawi, L. (V.) guyanensis, L. (V.) braziliensis and Endotrypanum sp. was detected. MAIN CONCLUSIONS In Brumptomyia sp. and Evandromyia termitophila, the first report of Leishmania DNA-detection is provided in Acre; Nyssomyia shawi is implicated as potential vector of L. (V.) braziliensis and L. (V.) guyanensis for the first time in Brazil.
Subject(s)
Animals , Female , Psychodidae/parasitology , DNA/analysis , Insect Vectors/genetics , Leishmania/genetics , Psychodidae/classification , Brazil , Polymerase Chain Reaction , DNA, Protozoan/analysis , Leishmaniasis, Cutaneous/transmission , Insect Vectors/classification , Insect Vectors/parasitology , Leishmania/isolation & purificationABSTRACT
OBJECTIVE: To develop a DNA detection kit for identification of Fetus Cervi, evaluate the quality indexes including specificity, stability, sensitivity and repeatability, and inspect the qualities of commercial Fetus Cervi samples. METHODS: The Fetus Cervi DNA test kit was developed and modified using the classical DNA extraction and PCR identification method. The genomic DNAs of Fetus Cervi and counterfeit goods were extracted by the kit and PCR technique was performed to identify the authenticity. The purity of DNA was detected by UV spectrophotometer. Finally, commercially available Fetus Cervi samples were verified. RESULTS: The kit was proved to be effective after 20 times of repeated frozen-thawing and it could be stored at - 20℃ for 1 year. The DNA could be detected when the primary solution was diluted by 200 times. The specificity test confirmed that the 15 samples were authentic Fetus Cervi, and 7 were counterfeit. The specificity of the kit was 100%. The repetitive experiments showed that the average intra-assay CV% and inter-assay CV% of the kit were 2.32% and 2.56%, respectively. CONCLUSION: The amount and purity of the nucleic acids extracted by the DNA detection kit can meet the requirement for identification of Fetus Cervi, and the kit has good specificity, high sensitivity and good stability, so it is suitable for the rapid detection of Fetus Cervi.
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OBJECTIVE: To establish a standard method for preserving the Fritillaria cirrhosa D. Don DNA fingerprint fragments. METHODS: Independently developed F. cirrhosa test kit was used to extract genomic DNA. Specific F. cirrhosa DNA fragments were cloned in vitro, and ligated into a carrier vector then transformed into a self replicating host cells to produce a large amount of specific F. Cirrhosa DNA. Plasmid DNA was extracted using Plasmid Extraction Kit and confirmed by PCR or restriction digestion of the insert. Bacterial colonies carrying authentic Sichuan F. cirrhosa specific DNA sequences were stored at -80℃. Stability was monitored at intervals of 1, 2, 3 and 6 months after DNA recovery using the boiling method of genomic DNA extraction. RESULTS: The amplified F. cirrhosa DNA clones showed clear bands in the electrophoresis, and had stable results in the repeated verification test. The amplified clones from resuscitated bacterial colonies which had been stored for 1, 2, 3, 6 months at -80℃ still displayed bright and clear bands after electrophoresis. CONCLUSION: It is feasible and effective to preserve DNA fingerprint of F. Cirrhosa by the established method, and this simple and reliable method can be used as the basis of establishing the new genes database of traditional Chinese medicine.
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OBJECTIVE: To develop a rapid DNA detection kit for DNA extraction and PCR identification of Panax ginseng C. A. Mey. METHODS: The classical DNA extraction and PCR identification methods for Panax ginseng C. A. Mey were modified, and the compositions and reaction conditions of the kit were determined. In addition, the specificity, stability, sensitivity, and repeatability of the kit were evaluated. The genomic DNAs of genuine and counterfeit ginseng goods were extracted by the kit and PCR was performed to identify the authenticity. The purity of the extracted DNA was detected by UV spectrophotometry. Finally, commercially available ginseng samples were verified. RESULTS: The purity of the genomic DNA extracted by the kit was (1.73 ± 0.13) (OD260/OD280), and a fragment between 150 and 200 bp could be amplified only from authentic Panax ginseng C. A. Mey. The specificity of the kit was 100%. The repetitive experiments showed that the average intra-assay CV% and inter-assay CV% of the kit were 2.38% and 2. 62%, respectively. The DNA in solutions diluted by 200 times could still be detected. Stability experiment proved that repeated freeze-thawing for 20 times had no significant effect on the activity of this kit and the test sample could be stored at - 20℃ for one year. The specificity test confirmed that 8 samples among the 10 commercial products were genuine, and 2 were counterfeit. CONCLUSION: The nucleic acid extraction and purity of the DNA detection kit can meet the requirement for identification of Panax ginseng C. A. Mey. The kit has good specificity, high sensitivity, and good stability, so it is suitable for the rapid detection of Panax ginseng C. A. Mey.
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Objective To analyze the differences of positive detection rate and copy number of human papillomavirus (HPV) DNA and E6/E7 mRNA between different grades of cervical lesions, and evaluate their clinical values in early screen?ing of cervical cancer. Methods The cervical exfoliated cell samples from 154 women undergoing biopsy examination and 32 objects undergoing hysterectomy (control group) were collected in Tianjin Central Hospital of Gynecology Obstetrics in 2014. According to the pathological results of cervical biopsy, 154 samples were divided into low-grade squamous intraepi?thelial lesion group (LSIL, n=51), high-grade squamous intraepithelial lesion group (HSIL, n=71), and squamous cell carci?noma group (SCC, n=32). HPV DNA was tested with hybrid capture technology, and E6/E7 mRNA was detected with fluores?cence quantitative hybridization. Immunohistochemistry was performed by detecting E6/E7 protein in all patients after sur?gery or cervical biopsy. Results Combined results of HPV DNA and E6/E7 mRNA demonstrated that the positive detection rate was significantly lower in control group than that of all levels of lesion groups (P 10 000 E6/E7 were significantly increased in high-grade squamous intraepithelial lesion group. Immunohistochemical results showed that the positive detection rate of E6/E7 was significantly lower in control group than that of all levels of lesion groups (P<0.05). The positive rate of E6/E7 was significantly higher in the high-grade squa?mous intraepithelial lesion group than that of low-grade group (P<0.05). Conclusion HPV infection is closely related to cervical abnormalities, which is one of effective measures for early screening of cervical cancer. The negative result of HPV DNA is very helpful to exclude the cervical abnormality, whereas the positive detection of mRNA has great value in predict?ing the disease. Combined results of positive detection and copy number make a comprehensive evaluation for the risk of cer?vical lesions.
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Magnetic fluorescent nanoparticles (Fe3O4/CdTe) were prepared in this work and applied for Toxoplasma gondii DNA detection. First, CdTe quantum dots were synthesized with 3- mercaptopropionic (MPA) capped. Fe3O4 magnetic particles were prepared by hydrothermal method with NaOH as precipitator, and they were surfacely modified with silane coupling agent (KH550). After then, the MPA-capped CdTe QDs were immobilized on the Fe3O4 particles surface via electrostatic interaction, and the Fe3O4/CdTe particles were prepared with the average size of 10 nm. The DNA sensing probe was fabricated through labeling a stem-loop Toxoplasma gondii DNA oligonucleotides with Fe3O4/CdTe (donor) at the 5′ end and BHQ2 (acceptor) at 3′ end, respectively. The assembly prosess was verified by UV-Vis, TEM, IR, XRD etc. The sensitivity characterization of the molecular beacon probe was performed by fluorescence spectrum (FS) with a detection limit of 8.339x10-9M. This chemical strategy can be further applied to prepare the magnetic nanoparticles for DNA detection.
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OBJECTIVE: To develop a kit for detection of DNA of Fritillaria cirrhosa D. Don and optimize its components as well as process protocols, in order to set up a simple, rapid molecular biology method for identification of Fritillaria cirrhosa D. Don. METHODS: All genomic DNA of Fritillaria cirrhosa D. Don was extracted by kit assay and pharmacopoeia method recorded in the expanded supplement of China Pharmacopoeia 2010, respectively; ultraviolet spectrophotometer was used to measure the quantity of extracted DNA; PCR amplification and restriction fragment length polymorphism analysis (RFLP) were carried out to identify the authentication of Fritillaria cirrhosa D. Don. RESULTS: The maximum value of genomic DNA extracted by pharmacopoeia method was (1.57±0.05) (OD260/OD280) and (1.73±0.10) by kit assay. The PCR amplification showed a single band over 300 bp, while the RFLP showed two distinct bands between 100 and 250 bp in agarose electrophoresis. CONCLUSION: The data demonstrated that the kit assay was better than the pharmacopoeia method, especially in the extraction quantity and DNA purity of Fritillaria cirrhosa D. Don nicleic acid; the PCR and RFLP results showed that the kit assay was consistent with pharmacopoeia method. The detection kit has good specificity, high sensitivity and good stability, so it is suitable for the rapid detection of Fritillaria cirrhosa D. Don.
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Cytomegalovirus (CMV) is a ubiquitous virus and its infections occur commonly after renal transplantation and immunosuppressive therapy. Early and accurate laboratory diagnosis of CMV infection in renal transplant is necessary but often difficult. To find optimal diagnostic methods for CMV infection, we compared shell vial culture and polymerase chain reaction (PCR) and Southern blot of PCR products. A total of 301 specimens of urine, blood neutrophils, tissues, or body fluids were obtained from 75 renal transplant recipients and were submitted to shell vial culture for CMV as well as DNA PCR using primers for immediate early(IE) gene of CMV. The human fibroblast cell line (MRC-5) was used to culture CMV and were examined with immunofluorescence staining using monoclonal antibody to the early antigen of CMV. The PCR products (274 and 379 bp) were detected by gel electrophoresis and ethidium bromide staining. When PCR products were not clearly visible on electrophoresis, PCR products were analyzed by Southern blot using IE gene probe. Sixty four(85.3%) of 75 renal transplant recipients showed CMV infection as analyzed by PCR and Southern blot as well as shell vial culture. On shell vial culture, CMV were detected in 81 specimens from 30(40%) renal transplant recipients in viremic state. On PCR and Southern blot analysis CMV were detected in 55 and 26 specimens, respectively from 59 patients. The sensitivity of culture and PCR to detect CMV infection were 42.4% and 83.3%, respectively. The results of two studies were concordant in 48%. PCR and Southern blot did not detect CMV in 10 and 5 culture proven CMV positive samples, respectively. Mutant CMV were found in 3 patients which showed 5-10 bp deletion in IE gene. Moreover, DNA sequencing analysis showed 5 mutant strains among 11 strains which appeared same by PCR prodcut. These results suggest that PCR followed by Southern blot may be more sensitive, but less specific than shell vial culture in the diagnosis of CMV disease. PCR followed by Southern blot may not detect mutant CMV. Combined analysis using both shell vial culture and PCR followed by Southern blot may be necessary to diagnose CMV infection in renal transplant recipients.
Subject(s)
Humans , Blotting, Southern , Body Fluids , Cell Line , Clinical Laboratory Techniques , Cytomegalovirus Infections , Cytomegalovirus , Diagnosis , DNA , Electrophoresis , Ethidium , Fibroblasts , Fluorescent Antibody Technique , Kidney Transplantation , Neutrophils , Polymerase Chain Reaction , Sequence Analysis, DNA , TransplantationABSTRACT
Objective To determine sequence of sporogony stage-specific (S type) 18S ribosomal RNA gene of Plasmodium yoelii (P.y) By265 strain, and by using it to detect the malaria parasites within vector mosquito. Methods A pair of conserved DNA primers, universe primer (Pu) and reverse transcription one (Pr), was designed and synthesized according to sequence of the 18S rRNA gene of Plasmodium berghei (P.b). The segment of the S type 18S rDNA of P.y was amplified by reverse transcript-polymerase chain reaction (RT-PCR) from dissected midguts of Anopheles stephensi infected with P.y on the 7th day after infective blood-meal, and its sequence was then determined. One P.y sporogony stage-specific primer (Pys) was selected according to the sequence. Using this primer and Pr, the parasites within mosquitoes were semi-quantitatively detected through RT-PCR between 1-7 d post-infection. Results The length of the amplified segment was 920 bp. Alignment in match region of the 18S rDNA among S type of P.y (PyS), S type of P.b (PbS) and asexual blood stage-specific one of P.y (PyA) revealed that the similarity between the former and the latter two reached 95^3% and 94^0% respectively. The density of amplified band was significantly concordance with the intensity of oocyst in the midgut. Sensitivity of RT-PCR method was higher than that of the traditional dissection and oocyst observation also. The assay could detect the 18S rRNA molecule of the parasites on the third day post-infection while their oocysts were difficult to be recognized under an optical microscope at that time. Conclusion This S type 18S rDNA sequence in P.y species was first reported (AF266261). As a molecular marker, it could be applied to monitoring the parasite development in its vector at an earlier stage semi-quantitatively with an adequate sensitivity and specificity.
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In order to investigate the possibility of analysis of single strand DNA by using DNA biosensor, we developed a quartz crystal DNA biosensor and studied its performances. The single strand DNA probe was immobilized on the surface of gold filled quartz crystal by biotin immunoreaction. The DNA biosensors were used to detect the complete complementary and single base mismatch complementary single strand DNA in solution. The DNA biosensors were regenerated with 0 1mol/L HCl after hybridization. The hybridization sensitivity of quartz crystal DNA biosensor is 10ng/ml. In the range of 5～40ng/ml, there was a linear correlation between responding signals and concentrations of complementary single strand DNA with a high specificity. The DNA biosensor could be repeatedly used over 5 times by using 0 1mol/L HCl for regeneration.