• Title/Summary/Keyword: Species-specific polymerase chain reaction

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Molecular Diagnosis of Streptococcus pneumoniae in Middle Ear Fluids from Children with Otitis Media with Effusion (삼출성 중이염 소아의 중이액에서 폐구균의 분자적 진단)

  • Byun, Sung Wan;Kim, Han Wool;Yoon, Seo Hee;Park, In Ho;Kim, Kyung-Hyo
    • Pediatric Infection and Vaccine
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    • v.22 no.2
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    • pp.106-112
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    • 2015
  • Purpose: The long-term administration of antibiotics interferes with bacterial culture in the middle ear fluids (MEFs) of young children with otitis media with effusion (OME). The purpose of this study is to determine whether molecular diagnostics can be used for rapid and direct detection of the bacterial pathogen in culture-negative MEFs. Methods: The specificity and sensitivity of both polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to the lytA gene of Streptococcus pneumoniae were comparatively tested and then applied for pneumococcal detection in the clinical MEFs. Results: The detection limit of the PCR assay was approximately $10^4$ colony forming units (CFU), whereas that of LAMP was less than 10 CFU for the detection of S. pneumoniae. Both PCR and LAMP did not amplify nucleic acid at over $10^6$ CFU of H. influenzae or M. catarrhalis, both of which were irrelevant bacterial species. Of 22 culture-negative MEFs from children with OME, LAMP positivity was found in twelve MEFs (54.5%, 12/22), only three of which were PCR-positive (25%, 3/12). Our results showed that the ability of LAMP to detect pneumococcal DNA is over four times higher than that of PCR (P<0.01). Conclusions: As a high-resolution tool able to detect nucleic acid levels equivalent to <10 CFU of S. pneumoniae in MEFs without any cross-reaction with other pathogens, lytA -specific LAMP may be applied for diagnosing pneumococcus infection in OME as well as evaluating the impact of a pneumococcal conjugate vaccine against OME.

Development and Validation of Multiplex Polymerase Chain Reaction to Determine Squid Species Based on 16s rRNA Gene (오징어류 종 판별을 위한 다중 유전자 검사법 개발 및 검증)

  • Kim, Hyunsu;Seo, Yong Bae;Choi, Seong-Seok;Kim, Jin-Hee;Shin, Jiyoung;Yang, Ji-Young;Kim, Gun-Do
    • Journal of Food Hygiene and Safety
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    • v.30 no.1
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    • pp.43-50
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    • 2015
  • In this study, single PCR and multiplex PCR tests were examined for identification of four types of squid species (giant squid, cuttlefish, octopus, beka squid) purchased from fish market as well as aquatic processed products in Busan. To design the specific primers against each species, the nucleotide sequences of the mitochondrial 16s rRNA gene of Architeuthis dux, Todarodes pacificus, Enteroctopus dofleini, Enteroctopus megalocyathus, Uroteuthis chinensis, Uroteuthis duvauceli, Uroteuthis edulis groups were analyzed for the identification of each species registered in the GeneBank (www.ncbi.nlm.nih.gov) and have been used for comparative analysis. In order to obtain the size variation of amplified fragments on multiplex PCR, we designed KOJ-F, OJ-F, OCT-F, HAN-F, ALLR primers for each species. The optimal PCR conditions and primers were selected for four types of squid species to determine target base sequences in its PCR products. In the case of single PCR, giant squid was only amplified by KOJ-F/ALLR primer; cuttlefish was only amplified by OJ-F/ALLR primer; octopus was only amplified by OCT-F/ALLR primer; and beka squid was only amplified by HAN-F/ALLR primer. For multiplex PCR, the mixture of four kinds of genomic DNA (giant squid, cuttlefish, octopus, beka squid) been prepared as a template and used together with the mixture of KOJ-F/OJ-F/OCT-F/HAN-F/ALLR primers in the reaction. By the multiplex PCR, it is confirmed that four samples are correspond to multiple simultaneous amplicon. Finally, we validated the established methods of multiplex PCR in the aquatic processed products. Although the mitochondrial 16s rRNA primers used in this study was useful as a marker for detection of each species among them, the study indicated that the established multiplex PCR method can be more useful tool for monitoring the processed products.

PREVALENCE OF STREPTOCOCCUS MUTANS AND STREPTOCOCCUS SOBRINUS IN CHILDREN WITH MIXED DENTITION (혼합치열기 어린이에서 Streptococcus mutans와 Streptococcus sobrinus의 분포도 조사)

  • Lee, Myung-Sung;Choi, Sung-Chul;Park, Jae-Hong
    • Journal of the korean academy of Pediatric Dentistry
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    • v.34 no.2
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    • pp.247-254
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    • 2007
  • Mutans streptococci have been reported to be implicated in dental caries. Of these streptococcal species, Streptcoccus mutans and Streptococcus sobrinus are most commonly found in human dental caries. Prevalence of these bacterial species in dental caries is found to be varied in different races and countries. Therefore, importance of these bacteria in dental caries remains to be determined The present study was performed to find out correlation S. mutans and S. sobrinus with dental caries in 125 Korean children with mixed dentition between 6 to 11 years of age. They were classified as group A(6-8 years) and group B(9-11 years) by age. For the study, stimulated saliva samples were collected from each subject. The vials containing saliva specimens were serially diluted (1:10) in saline and plated in duplicate on tryptone-yeast extract-cysteine with sucrose and bacitracin (TYCSB) for S. mutans and S. sobrinus. After genomic DNA was extracted from the samples, polymerase chain reaction (PCR) amplification was performed for identification using universal primers and specific primers to S. mutans and S. sobrinus. Data of microbial variables were compared to caries status of the subjects. According to this study, the result were as follows : 1. S. mutans versus S. sobrinus were moderate positive linear correlated in both group A(r=0.70) and group B(r=0.50). 2. Between S. mutans and dental caries there were weak positive linear correlation in both group A(r= 0.25) and group B(r=0.34). 3. S. sobrinus versus dental caries were not correlated in group A but slightly correlated in group B(r=0.21). 4. Between S. mutans and age, there were not correlation in both group. 5. S. sobrinus versus age were weak correlated in group A(r=0.32) but not correlated in group B.

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Detection and Identification of Mycobacterium Tuberculosis in Patients with Tuberculous Cervical Lymphadenitis by PCR-RFLP (경부 결핵성 임파선염 환자에서 PCR-RELP를 이용한 결핵균의 검출 및 확인)

  • Lee Sang-Sook;Cho Young-Rok;Chun Ji-Min;Choi Yong-Seok;Sohn Eun-Ju;Park Nam-Cho;Park June-Sik
    • Korean Journal of Head & Neck Oncology
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    • v.12 no.2
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    • pp.169-176
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    • 1996
  • Tuberculous cervical lymphadenitis is still an important cause of neck mass in Korea. Tuberculosis is an important differential diagnosis in patients of cervical lymphadenopathy. Rapid and sensitive test for the diagnosis of tuberculosis is essential for the approapiate treatment. Up to now, conventional diagnostic methods for M. tuberculosis were acid-fast bacilli(AFB) stain and culture of M. tuberculosis. The direct microscopic examination of AFB by Ziehl-Neelsen stain is rapid, but often negative. The culture for M. tuberculosis is time-consuming, taking 4 to 8 weeks. Recently various methods to detect Mycobacterial DNA, including PCR and restriction fragment length polymorphism(RFLP) analysis have been reported. Here we represent a simple method for the confirmation of M. tuberculosis and exclusion of the other Mycobacterial species by RFLP analysis and silver staining of polyacrylamide gel electrophoresis after nested PCR for a repetitive DNA sequence(IS986) specific for M. tuberculosis from fresh or paraffin-embedded biopsy specimens. This result leads us to conclude that this method is simple, rapid and possibly applicable to confirm M. tuberculosis and rule out the other Mycobacteria species from the clinical specimens in the clinical laboratories.

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Detection Method for Identification of Pueraria mirifica (Thai kudzu) in Processed Foods (가공식품 중 태국칡(Pueraria mirifica) 혼입 판별법 개발)

  • Park, Yong-Chjun;Jin, Sang-Wook;Kim, Mi-Ra;Kim, Kyu-Heon;Lee, Jae-Hwang;Cho, Tae-Yong;Lee, Hwa-Jung;Lee, Sang-Jae;Han, Sang-Bae
    • Journal of Food Hygiene and Safety
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    • v.27 no.4
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    • pp.466-472
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    • 2012
  • In this study, ribulose bisphosphate carboxylase (rbcL), RNApolymeraseC (rpoC1), intergenic spacer (psbA-trnH), and second internal transcribed spacer (ITS2) as identification markers for discrimination of P. mirifica in foods were selected. To be primer design, we obtained 719 bp, 520 bp, 348 bp, and 507 bp amplicon using universal primers from selected regions of P. mirifica. The regions of rbcL, rpoC1, and psbA-trnH were not proper for design primers because of high homology about P. mirifica, P. lobata, and B. superba. But, we had designed 4 pairs of oligonucleotide primers from ITS2 gene. Predicted amplicon from P. mirifica were obtained 137 bp and 216 bp using finally designed primers SFI12-miri-6F/SFI12-miri-7R and SFI12-miri-6F/SFI12-miri-8R, respectively. The species-specific primers distinguished P. mirifica from related species were able to apply food materials and processed foods. The developed PCR method would be applicable to food safety management for illegally distributed products in markets and internet shopping malls.

Genetic Variation of Mitochondrial DNA in Duroc (Sus Scrofa) Using Single Stranded Conformation Polymorphism Analysis (Single Stranded Conformation Polymorphism 분석에 의한 돼지 Duroc 품종의 미토콘드리아 DNA 유전적 변이)

  • Cho, I.C.;Jung, Y.H.;Jung, J.K.;Seong, P.N.;Kim, B.W.;Lee, J.G.;Jeon, J.T.
    • Journal of Animal Science and Technology
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    • v.45 no.6
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    • pp.911-916
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    • 2003
  • The mitochondrial DNA(mtDNA) D-loop region was amplified from Duroc(Sus scrofa) by polymerase chain reaction(PCR). The oligonucleotide primer used to amplify the Sus scrofa mtDNA D-loop region was designed using tRNA-Pro and tRNA-Phe sequence in mtDNA regions highly conserved in many other animal species. There were 1,145 base pairs(bp) in the D-loop region. The middle of the region contained 10 tandem repeat of an 10-bp Sus scrofa-specific sequence, TACACGTGCG. We designed primers for PCR-mediated single stranded conformation polymorphism(SSCP) analysis that amplified a 345 bp fragment, which contained the most variable region according to our sequencing data. SSCP analysis of denatured amplification products was carried out by polyacrylamide(8%) gel electrophoresis followed by ethidium bromide staining. The SSCP analysis identified two band patterns(A and B) and comparision of these two nucleotide sequences identified 21 base substitutions. These results show that SSCP analysis of the D-loop region is useful for detecting the genetic polymorphism.

Trends in the rapid detection of infective oral diseases

  • Ran-Yi Jin;Han-gyoul Cho;Seung-Ho Ohk
    • International Journal of Oral Biology
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    • v.48 no.2
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    • pp.9-18
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    • 2023
  • The rapid detection of bacteria in the oral cavity, its species identification, and bacterial count determination are important to diagnose oral diseases caused by pathogenic bacteria. The existing clinical microbial diagnosis methods are time-consuming as they involve observing patients' samples under a microscope or culturing and confirming bacteria using polymerase chain reaction (PCR) kits, making the process complex. Therefore, it is required to analyze the development status of substances and systems that can rapidly detect and analyze pathogenic microorganisms in the oral cavity. With research advancements, a close relationship between oral and systemic diseases has been identified, making it crucial to identify the changes in the oral cavity bacterial composition. Additionally, an early and accurate diagnosis is essential for better prognosis in periodontal disease. However, most periodontal disease-causing pathogens are anaerobic bacteria, which are difficult to identify using conventional bacterial culture methods. Further, the existing PCR method takes a long time to detect and involves complicated stages. Therefore, to address these challenges, the concept of point-of-care (PoC) has emerged, leading to the study and implementation of various chair-side test methods. This study aims to investigate the different PoC diagnostic methods introduced thus far for identifying pathogenic microorganisms in the oral cavity. These are classified into three categories: 1) microbiological tests, 2) microchemical tests, and 3) genetic tests. The microbiological tests are used to determine the presence or absence of representative causative bacteria of periodontal diseases, such as A. actinomycetemcomitans, P. gingivalis, P. intermedia, and T. denticola. However, the quantitative analysis remains impossible, and detecting pathogens other than the specific ones is challenging. The microchemical tests determine the activity of inflammation or disease by measuring the levels of biomarkers present in the oral cavity. Although this diagnostic method is based on increase in the specific biomarkers proportional to inflammation or disease progression in the oral cavity, its commercialization is limited due to low sensitivity and specificity. The genetic tests are based on the concept that differences in disease vulnerability and treatment response are caused by the patient's DNA predisposition. Specifically, the IL-1 gene is used in such tests. PoC diagnostic methods developed to date serve as supplementary diagnostic methods and tools for patient education, in addition to existing diagnostic methods, although they have limitations in diagnosing oral diseases alone. Research on various PoC test methods that can analyze and manage the oral cavity bacterial composition is expected to become more active, aligning with the shift from treatment-oriented to prevention-oriented approaches in healthcare.

Detection and Differentiation of Intentional and Unintentional Mixture in Raw Meats Using Real-time PCR (Real-time PCR을 이용한 식육원료의 의도적, 비의도적 혼입 판별법 개발)

  • Kim, Kyu-Heon;Kim, Mi-Ra;Park, Young-Eun;Kim, Yong-Sang;Lee, Ho-Yeon;Park, Yong-Chjun;Kim, Sang Yub;Choi, Jang Duck;Jang, Young-Mi
    • Journal of Food Hygiene and Safety
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    • v.29 no.4
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    • pp.340-346
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    • 2014
  • In this study, the detection method was developed using real-time PCR to distinguish 4 species (bovine, porcine, horse, and chicken) of raw meats. The genes for distinction of species about meats targeted at 12S rRNA and 16S rRNA parts in mitochondrial DNA. Probes were designed to have a 5' FAM and a TAMRA at the 3' end. This study is to develop 4 species-specific primer and probes about raw materials and real-time PCR on 10 strains to observe the products of non-specific signal for similar species. As a result, any non-specific signal were not detected among each other. Real-time PCR method was developed for quantitation and identification of intentional and unintentional mixture in ground mixed meat (The difference of $C_T$ value between intentional mixture and 100% meat: $${\leq_-}$$ cycles, The difference of $C_T$ value between unintentional mixture and 100% meat: $${\geq_-}$$ cycles). The detection and differentiation of intentional and unintentional mixture in this study would be applied to food safety management for eradication of adulterated food distribution and protection of consumer's right.

Effects of Estrogen on the Transcriptional Activities of Catecholamine Biosynthesizing Enzymes in the Brain and Adrenal Gland of Ovariectomized Rats (난소 절제 흰쥐의 뇌와 부신에서의 Catecholamine Biosynthesizing Enzyme들의 전사에 미치는 Estrogen의 효과)

  • 유경신;이종화;최돈찬;이성호
    • Development and Reproduction
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    • v.6 no.2
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    • pp.117-122
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    • 2002
  • Dopamine(DA), norepinephrine(NE), and epinephrine(E) belong to a class of neurotransmitters known as catecholamine (CA) which are synthesized and secreted by mammalian brain and adrenal medulla. CA regulate several behavior patterns connected with breeding, and regulate GnRH-gonadotropin hormone axis' vitality between hypothalamus-pituitary gland linking with reproduction freeze. The present study examined effects of sex steroid hormone on the transcriptional activities of CA biosynthesis enzymes, tyrosine hydroxylase(TH), dopamine $\beta$ -hydroxylase(DBH), and phenylethaolamine-N-methyl transferase(PNMT). Mature female rats were ovariectomized(OVX) and implanted with 17 $\beta$-estradiol(E$_2$: 500 $\mu\textrm{g}$/ml) or sesame oil. Forty-eight hours after implantation all the animals were sacrificed. Total RNAs were extracted immediately and were applied to semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR). The expression level of TH was appeared by hypothalamus > SNc> adrenal medulla orders in OVX+Oil group, and by SNc > hypothalamus) adrenal medulla orders in OVX+E$_2$ group. Treatment with E$_2$ significantly increased TH expression in SNc and adrenal medulla but in hypothalamus, the reduced TH expression was observed. The expression level of DBH was appeared by adrenal medulla > SNc > hypothalamus orders in OVX+Oil group and in OVX+E$_2$ group. Administration of E$_2$ significantly reduced DBH expression in SNc, and increased in adrenal medulla. Two cDNA products, large(PNMT1) and small(PMNTs) species of 110bp difference, were amplified in SNc and hypothalamus, but only PNMTs was observed in adenal medulla. The PNMTs expression level was in the order of adrenal medulla > hypothalamus > SNc in both OVX+Oil and OVX+E$_2$ group. The PNMTs expression in SNc and adrenal medulla was significantly increased byE$_2$. The present report demonstrated that estrogen effects on transcriptional activities for CA biosynthethic enzymes were tissue specific in adrenal medulla as well as different region of brain. These results suggest that it might be crucial relationship between the type of estrogen receptor and CA enzyme gene expression.

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Detection of Campylobacter jejuni in food and poultry visors using immunomagnetic separation and microtitre hybridization

  • Simard, Ronald-E.
    • Proceedings of the Korean Society of Fisheries Technology Conference
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    • 2000.05a
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    • pp.71-73
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    • 2000
  • Campylobacter jejuni is most frequently identified cause of cause of acute diarrhoeal infections in developeed countries, exceeding rates of illness caused by both salmonella and shigilla(Skirrow, 1990 ; Lior 1994). Previous studies on campylobacter jejuni contamination of commercial broiler carcasses in u.s.(Stern, 1992). Most cases of the disease result from indirect transmission of Campylobactor from animals via milk, water and meat. In addition to Campylobactor jejuni. the closely relates species Campylobactor coli and Campylobactor lari have also been implicated as agents of gastroenteritis in humans. Campylobactor coli represented only approximately 3% of the Campylobactor isolates from patients with Campylobactor enteritis(Griffiths and Park, 1990) whereas Campylobactor coli is mainly isolated from pork(Lmmerding et al., 1988). Campylobactor jejuni has also been isolated from cases of bacteremia, appendicitis and, recently, has been associated with Guillai-Barre syndrome(Allos and Blaser, 1994; von Wulffen et al., 1994; Phillips, 1995). Studies in volunteers indicated that the infectious dose for Campylobactor jejuni is low(about 500 organisms)(Robinson, 1981). The methods traditionally used to detect Campylobactor ssp. in food require at least two days of incubation in an enrichment broth followed by plating and two days of incubation on complex culture media containing many antibiotics(Goossens and Butzler, 1992). Finnaly, several biochemical tests must be done to confirm the indentification at the species level. Therfore, sensitive and specific methods for the detection of small numbers of Campylobactor cells in food are needed. Polymerase chain reaction(PCR) assays targeting specific DNA sequences have been developed for the detection of Campylobactor(Giesendorf and Quint, 1995; Hemandex et al., 1995; Winter and Slavidk, 1995). In most cases, a short enrichment step is needed to enhance the sensitivity of the assay prior to detection by PCR as the number of bacteria in the food products is low in comparison with those found in dinical samples, and because the complex composition of food matrices can hinder the PCR and lower its sensitivity. However, these PCR systems are technically demanding to carry out and cumbersome when processing a large number of samples simutaneously. In this paper, an immunomagnetic method to concentrate Campylobactor cells present in food or clinical samples after an enrichment step is described. To detect specifically the thermophilic Campylobactor. a monoclonal antibody was adsorbed on the surface of the magnetic beads which react against a major porin of 45kDa present on the surface of the cells(Huyer et al., 1986). After this partial purification and concentration step, detection of bound cells was achieved using a simple, inexpensive microtitre plate-based hybridization system. We examined two alternative detection systems, one specific for thermophilic Campylobactor based on the detection of 23S rRNA using an immobilized DNA probe. The second system is less specific but more sensitive because of the high copy number of the rRNA present in bacterial cell($10^3-10^4$). By using specific immunomagnetic beads against thermophilic Campylobactor, it was possible to concentrate these cells from a heterogeneous media and obtain highly specific hybridization reactions with good sensitivity. There are several advantages in using microtitre plates instead of filter membranes or other matrices for hybridization techniques. Microtitre plates are much easier to handle than filter membranes during the adsorption, washing, hybridization and detection steps, and their use faciilitates the simultanuous analysis of multiple sample. Here we report on the use of a very simple detection procedure based on a monoclonal anti-RNA-DNA hybrid antibody(Fliss et al., 1999) for detection of the RNA-DNA hybrids formed in the wells.

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