• Parasites, Hosts and Diseases
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• v.53 no.2
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• pp.147-154
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• 2015

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (${\approx}375bp$) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ${\approx}550bp$ of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ${\approx}2$ oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

• Fisheries and Aquatic Sciences
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• v.11 no.4
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• pp.205-208
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• 2008

The mixotrophic dinoflagellate Cochlodinium polykrikoides was reported to be linked to major fish kills in Korea and Japan since the 1990s. Rapid and sensitive detection of microalgae has been problematic because morphological identification of dinoflagellates requires light microscopic and scanning electron microscopic observations that are time consuming and laborious compared to real-time PCR. To address this issue, a real-time PCR probe targeting the ITS2 rRNA gene was used for rapid detection and quantification of C. polykrikoides. PCR inhibitors in water column samples were removed by dilution of template DNA for elimination of false-negative reactions. A strong association between cell quantification using real-time PCR and microscopic counts suggests that the real-time PCR assay is an alternative method for cell estimation of C. polykrikoides in environment samples.

• Biomedical Science Letters
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• v.25 no.1
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• pp.15-22
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• 2019

The diagnosis of atopic dermatitis (AD) includes a test that checks allergen-mediated skin reactions and a method of measuring the total IgE and allergen-specific IgE in blood. However, these test methods are performed directly on the patient, which cause some pain or discomfort. In addition, the skin response test or IgE may result in false negative in about 20% of patients. In the present study, to identify specific biomarkers, HaCaT cells were used as a human keratinocyte that make up the skin, were treated IL-4 and IL-13 for 24 hours to induce a situation similar to keratinocytes in AD patients. In the HaCaT cells, pro-inflammatory cytokine such as IL-5, IL-6, and MCP-1 were increased by IL-4 and IL-13 and skin barrier proteins was reduced by IL-4 and L-13. This results showed that a situation similar to the stratum corneum of an actual patient is induced in HaCaT cells. And then the secretions of Kallikrein (KLK) 5 and KLK7 protease were checked by enzyme-linked immunosorbent assay (ELISA). It was specifically increased by IL-4 and IL-13. This showed that AD-related protease can be detected at the protein level using keratinocytes that can be taken in a non-invasive manner and suggested the possibility of applying it to AD diagnosis.

• Parasites, Hosts and Diseases
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• v.24 no.1
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• pp.25-41
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• 1986

The applicability of micro-ELISA was evaluated in human neuro-cysticercosis using paired samples of serum and CSF. A total of 355 cases who were mostly neurologic patients was subjected. Cystic fluid of C. cellulosae was used as antigen in protein concentration of $2.5{\;}{\mu}g/ml$. Serum was diluted to 1 : 100 and CSF was undiluted in the assay for the specific IgG antibody level. The differential criterion of the positive reaction was the abs. of o. 18 in both samples. The results were summarized as follows: 1. The overall sensitivity of the micro-ELISA in 71 confirmed neurocysticercosis was 90.1% ; the sensitivity by serum was 77.5% and that by CSF was 83.1%. CSF was a more sensitive and valuable material. Most of the false negative cases of neuro-cysticercosis showed far lower level of abs. rather than marginal. 2. The overall specificity of the micro-ELISA in 52 confirmed other neurologic diseases was 88.5%; the specificities by serum and by CSF were 94.2% respectively. Cases of other neurologic diseases did not show false positive reactions in both samples. 3. When serum was assayed, taeniasis(2/18), sparganosis(2/20), paragonimiasis(1/56), clonorchiasis(1/15) and fascioliasis(1/1) cases showed cross reactions. When CSF was assayed, 2 ot 10 neuro-sparganosis showed cross reactions while none of 9 neuro-paragonimiasis showed it. Out of 71 confirmed neuro-cysticercosis cases, 6 and 11 showed cross reactions by serum and CSF to crude extract antigen of sparganum; but no case did show it to crude extract antigen of Paragonimus westermani. 4. Ventricular CSF showed low or negative levels of IgG antibody than lumbar CSF unless the lesion was at the lateral ventricle itself. 5. Out of 4 racemose cysticercosis cases, 3 showed positive reaction in serum while all of 3 examined CSF were positive. The above results indicated that the serological test for detecting the specific IgG antibody by micro-ELISA using paired samples of serum and CSF was very helpful for clinical differentiation of neuro-cysticercosis from neurologic diseases of other causes.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.48 no.2
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• pp.158-167
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• 2015

A lateral flow immunoassay kit based on antigen-antibody interactions was developed to detect residues of beta-lactams, quinolones, tetracyclines, and sulfonamides in farmed fish. Group-specific antibodies showing cross-reactivity with other antibiotics in the same group were produced in rabbits. The rabbits were immunized eight times to obtain the maximum titers. Antibodies were extracted from the antisera collected from the immunized rabbits and produced group-specific reactions with antibiotics from the four groups. A kit was prepared that optimize conditions for the antigen-antibody reaction, using colloidal gold conjugated antibodies, and was designed to detect the four groups of antibiotics simultaneously. The kit enabled the detection of antibiotics in the four groups at below maximum residue limits (MRLs), which were $200{\mu}g/kg$ for tetracyclines, $100{\mu}g/kg$ for sulfonamides, $50{\mu}g/kg$ for beta-lactams, and $100{\mu}g/kg$ for quinolones. The cross-reactivity of the antibodies ranged from 10-80% for the sulfonamides, 20-100% for tetracyclines, 38-100% for quinolones, and 20-100% for the beta-lactams, confirming that the antibodies were group specific. The test kit was used 30 times to examine spiked antibiotics at the limits of detection (LODs) and all produced positive results, indicating high sensitivity. The LODs for the assay ranged from 4-20 ng/mL for beta-lactams, 25-50 ng/mL for sulfonamides, 20-100 ng/mL for tetracyclines, and 30-80 ng/mL for quinolones, and there were no false negative reactions at above these LODs. In addition, all of the LODs of the developed kit were correlated with high-performance liquid chromatography (HPLC) data. Our lateral flow immunoassay kit can simultaneously detect antibiotic residues from a large number of fish samples rapidly, strengthening the safety of domestic farmed and imported fish.

• Annals of Clinical Microbiology
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• v.22 no.1
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• pp.9-13
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• 2019

Background: The isolation of carbapenemase-producing Enterobacteriaceae (CPE) has become increasingly common. Continuous surveillance for these organisms is essential because their infections are closely related to outbreaks of illness and are associated with high mortality rates. The aim of this study was to develop and evaluate multiplex PCR as a means of detecting several important CPE genes simultaneously. Methods: We aimed to develop a multiplex PCR that could detect seven CPE genes simultaneously. The multiplex PCR was composed of seven primer sets for the detection of KPC, IMP, VIM, NDM-1, GES, OXA-23, and OXA-48. We designed different PCR product sizes of at least 100 bp. We evaluated the performance of this new test using 69 CPE-positive clinical isolates. Also, we confirmed the specificity to rule out false-positive reactions by using 71 carbapenem-susceptible clinical strains. Results: A total of 69 CPE clinical isolates showed positive results and were correctly identified as KPC (N=14), IMP (N=13), OXA-23 (N=12), OXA-48 (N=11), VIM (N=9), GES (N=5), and NDM (N=5) by the multiplex PCR. All 71 carbapenem-susceptible clinical isolates, including Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, showed negative results. Conclusion: This multiplex PCR can detect seven CPE genes at a time and will be useful in clinical laboratories.