Abstract:In domestic animals of the family Bovidae, sex preselection of offspring has been
demanded for convenience of milk/beef production and animal breeding. Development of the
nonsurgical embryo transfer technique and sexing methods of preimplantation embryos made
it possible. Sexing based on detection of Y chromosome-specific DNA sequences is
considered the most reliable method to date. PCR enables amplification of a target
sequence from a small number of blastomeres. However, it requires technical skill and is
ti… Show more
“…Furthermore, LAMP is less influenced by inhibitors present in complex samples than standard PCR, which is highly beneficial for clinical specimens such as blood components, sputum, feces, or body fluids ( Kaneko et al, 2007 ). LAMP assays have been widely applied to genetic diagnoses, the detection of epidemic bacteria ( Hara-Kudo et al, 2005 ; Song et al, 2005 ) and viruses ( Okafuji et al, 2005 ), fetal sex identification ( Hirayama et al, 2013 ), and parasite recognition ( Chen et al, 2011 ; Kong et al, 2012 ).…”
Ebola virus (EBOV) can lead to severe hemorrhagic fever with a high risk of death in humans and other primates. To guide treatment and prevent spread of the viral infection, a rapid and sensitive detection method is required for clinical samples. Here, we described and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect Zaire ebolavirus using the nucleoprotein gene (NP) as a target sequence. Two different techniques were used, a calcein/Mn2+ complex chromogenic method and real-time turbidity monitoring. The RT-LAMP assay detected the NP target sequence with a limit of 4.56 copies/μL within 45 min under 61°C, a similar even or increase in sensitivity than that of real-time reverse transcription-polymerase chain reaction (RT-PCR). Additionally, all pseudoviral particles or non- Zaire EBOV genomes were negative for LAMP detection, indicating that the assay was highly specific for EBOV. To appraise the availability of the RT-LAMP method for use in clinical diagnosis of EBOV, of 417 blood or swab samples collected from patients with clinically suspected infections in Sierra Leone, 307 were identified for RT-LAMP-based surveillance of EBOV. Therefore, the highly specific and sensitive RT-LAMP method allows the rapid detection of EBOV, and is a suitable tool for clinical screening, diagnosis, and primary quarantine purposes.
“…Furthermore, LAMP is less influenced by inhibitors present in complex samples than standard PCR, which is highly beneficial for clinical specimens such as blood components, sputum, feces, or body fluids ( Kaneko et al, 2007 ). LAMP assays have been widely applied to genetic diagnoses, the detection of epidemic bacteria ( Hara-Kudo et al, 2005 ; Song et al, 2005 ) and viruses ( Okafuji et al, 2005 ), fetal sex identification ( Hirayama et al, 2013 ), and parasite recognition ( Chen et al, 2011 ; Kong et al, 2012 ).…”
Ebola virus (EBOV) can lead to severe hemorrhagic fever with a high risk of death in humans and other primates. To guide treatment and prevent spread of the viral infection, a rapid and sensitive detection method is required for clinical samples. Here, we described and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect Zaire ebolavirus using the nucleoprotein gene (NP) as a target sequence. Two different techniques were used, a calcein/Mn2+ complex chromogenic method and real-time turbidity monitoring. The RT-LAMP assay detected the NP target sequence with a limit of 4.56 copies/μL within 45 min under 61°C, a similar even or increase in sensitivity than that of real-time reverse transcription-polymerase chain reaction (RT-PCR). Additionally, all pseudoviral particles or non- Zaire EBOV genomes were negative for LAMP detection, indicating that the assay was highly specific for EBOV. To appraise the availability of the RT-LAMP method for use in clinical diagnosis of EBOV, of 417 blood or swab samples collected from patients with clinically suspected infections in Sierra Leone, 307 were identified for RT-LAMP-based surveillance of EBOV. Therefore, the highly specific and sensitive RT-LAMP method allows the rapid detection of EBOV, and is a suitable tool for clinical screening, diagnosis, and primary quarantine purposes.
“…The LAMP assay does not require skilled operators or precise instruments, and is therefore practically applicable within point of care settings with poor resources. Since its introduction by Notomi et al ( 8 ) in 2000, it has been widely applied to a variety of fields, including clinical diagnosis, food safety, livestock breeding and determination of sex ( 18 , 19 ). This is largely due to its rapid and simple methodology.…”
The binary toxin Clostridium difficile transferase (CDT) is frequently observed in C. difficile strains and is associated with an increased severity of C. difficile infection. CDT-producing C. difficile infections cause higher fatality rates than infections with CDT negative isolates. Thus, the rapid and accurate identification of a CDT positive C. difficile infection is critical for effective treatment. The present study demonstrates how loop-mediated isothermal amplification (LAMP) can be used to detect CDT-producing C. difficile based on visual observation. This is a low complexity, rapid molecular method that has the potential to be used within a point of care setting. The specificity and sensitivity of the primers in the LAMP reactions for CDT detection were determined using two different methods, a real-time turbidity monitor and visual detection after the addition of calcein to the reaction tube. The results revealed that target DNA was amplified and visualized by these two detection methods within 60 min at a temperature of 60°C. The sensitivity of the LAMP assay was identified to be 10-fold greater than that of polymerase chain reaction analysis. When 25 alternative bacterial strains lacking CDT were tested, the results of the amplification were negative, confirming the specificity of the primers. In conclusion, the visual LAMP method established in the present study may be a rapid, reliable and cost-effective tool for detecting CDT-producing C. difficile strains at the point of care.
“…We showed that both genomic DNA a RNA can be isolated from single blastocysts, however due to the little amount of resultant DNA, we recommend improving the protocol of amplification of such minute amount of genomic DNA to give satisfactory results. Other researchers improved the DNA amplification used different kind of PCR protocol such as loop-mediated isothermal amplification (LAMP) [25], [26].Fig. 1Representative image of gel electrophoresis showing validation of amplification of Y-specific segment by PCR.…”
Single embryo culture is essential for culturing embryos derived from few oocytes obtained from elite cows through ultrasonography guidance. Bovine in vitro fertilization (IVF) and individual embryo culture is a challenge as it generally leads to impaired embryo development. In this study, we explored the embryonic development and the sex ratio of IVF-derived bovine embryo cultured individually in chemically defined two-step culture medium. Total 63 cumulus-oocyte complexes were collected, in vitro matured, in vitro fertilized and the resultant fertilized oocytes were randomly cultured individually (4 trials, 15–16 oocytes each) in microdrops of 5 µL of a chemically defined two-step culture medium. Blastocysts were counted in every trial (n = 32, 50.79%) and all of them were used for both genomic DNA and total RNA extraction, cDNA synthesis and PCR using specific primers for GAPDH, GDP6, XIST and SRY genes. Results showed significant difference in expression of XIST (positive expression in 11 blastocysts) and SRY (positive expression in 21 blastocysts) mRNAs, P < .05. This result supports the hypothesis of sexual dimorphism among the pre-implantation in vitro produced embryos and provides an efficient medium for single bovine embryos in vitro production.
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