임상미생물 검출을 위한 광대한 범위와 특이도를 가지는 16S rRNA PCR법 개발 Development of Broad-range and Specific 16S rRNA PCR for Use in Routine Diagnostic Clinical Microbiology원문보기
16S rRNA gene PCR법은 환자 검체로부터 병원성 미생물을 검출 및 동정에 사용되어진다. 본 연구는 대량의 임상미생물 진단을 위해 bacterial 16S rRNA 부위 유전자 서열을 이용하여 광대한 범위와 높은 특이도를 가지는 primer을 포함한 PCR법을 개발하였다. 10개 표준 균주 16S rRNA 보존 부위의 유전자 서열을 기반으로 primer set를 구축하였다. 98명 환자 검체에서 임상 미생물을 분리하였다. 98개 균주는 phenotypic 방법을 이용하여 확인하고, 개발된 primer set와 universal primer set를 이용한 PCR법으로 확인하였다. 획득한 PCR 산물은 forward primer, reverse primer, 그리고 자동화 DNA 분석기를 이용하여 각 균주의 16S rRNA 유전자 서열을 분석 및 확인하였다. 본 연구에서 개발된 primer set와 universal primer set의 임상미생물 검출에 대한 효율성을 평가하였고, 또한 phenotypic 방법과 분자생물학적 방법을 비교했다. 분리된 98개 균주를 대상으로 개발된 primer set로 16S rRNA PCR을 진행하여 778 bp 크기의 단일밴드로 증폭 되었음을 확인했다. 총 98개중 94개 균주(95.9%)는 phenotypic 결과와 동일함을 확인했다. 새로 개발된 primer set를 이용한 결과는 universal primer set를 이용한 98개 균주(100%)의 결과와 동일함을 확인하였다. 개발된 16S rRNA gene PCR법은 임상미생물 검출 및 동정에서 신속성, 정확성, 그리고 검사 비용 절감의 장점을 가진다. 개발된 primer set는 병원성 미생물 동정에서 효율성을 확인했다.
16S rRNA gene PCR법은 환자 검체로부터 병원성 미생물을 검출 및 동정에 사용되어진다. 본 연구는 대량의 임상미생물 진단을 위해 bacterial 16S rRNA 부위 유전자 서열을 이용하여 광대한 범위와 높은 특이도를 가지는 primer을 포함한 PCR법을 개발하였다. 10개 표준 균주 16S rRNA 보존 부위의 유전자 서열을 기반으로 primer set를 구축하였다. 98명 환자 검체에서 임상 미생물을 분리하였다. 98개 균주는 phenotypic 방법을 이용하여 확인하고, 개발된 primer set와 universal primer set를 이용한 PCR법으로 확인하였다. 획득한 PCR 산물은 forward primer, reverse primer, 그리고 자동화 DNA 분석기를 이용하여 각 균주의 16S rRNA 유전자 서열을 분석 및 확인하였다. 본 연구에서 개발된 primer set와 universal primer set의 임상미생물 검출에 대한 효율성을 평가하였고, 또한 phenotypic 방법과 분자생물학적 방법을 비교했다. 분리된 98개 균주를 대상으로 개발된 primer set로 16S rRNA PCR을 진행하여 778 bp 크기의 단일밴드로 증폭 되었음을 확인했다. 총 98개중 94개 균주(95.9%)는 phenotypic 결과와 동일함을 확인했다. 새로 개발된 primer set를 이용한 결과는 universal primer set를 이용한 98개 균주(100%)의 결과와 동일함을 확인하였다. 개발된 16S rRNA gene PCR법은 임상미생물 검출 및 동정에서 신속성, 정확성, 그리고 검사 비용 절감의 장점을 가진다. 개발된 primer set는 병원성 미생물 동정에서 효율성을 확인했다.
Broad-range and specific 16S rRNA gene PCR is used for detection and identification of bacterial pathogens in clinical specimens from patients with a high suspicion for infection. We describe the development of a broad-range and specific PCR primer, based on bacterial 16S rRNA, for use in routine di...
Broad-range and specific 16S rRNA gene PCR is used for detection and identification of bacterial pathogens in clinical specimens from patients with a high suspicion for infection. We describe the development of a broad-range and specific PCR primer, based on bacterial 16S rRNA, for use in routine diagnostic clinical microbiology services. The primers were designed by using conservative regions of 16S rRNA sequences from 10 strains. Ninety-eight clinical strains were isolated from clinical patient specimens. A total of 98 strains of bacteria were identified by phenotypic methods; PCR with newly designed primers and universal primers. All purified PCR products were sequenced using both forward and reverse primers on an automated DNA analyzer. In this study, we evaluated the usefulness of the newly designed primers and the universal primers for the detection of bacteria, and both these techniques were compared with phenotypic methods for bacteria detection. When we also tested 98 strains of clinical isolates with newly designed primers, about 778 bp DNA fragments were amplified and identified from all strains. Of the 98 strains, 94 strains (95.9%) correspond in comparison with phenotypic methods. The newly designed primers showed that the identities of 98 (100%) strains were the same as those obtained by universal PCR primers. The overall agreement between the newly designed primers and universal primers was 100%. The primer set was designed for rapid, accurate, and cheap identification of bacterial pathogens. We think the newly designed primer set is useful for the identification of pathogenic bacteria.
Broad-range and specific 16S rRNA gene PCR is used for detection and identification of bacterial pathogens in clinical specimens from patients with a high suspicion for infection. We describe the development of a broad-range and specific PCR primer, based on bacterial 16S rRNA, for use in routine diagnostic clinical microbiology services. The primers were designed by using conservative regions of 16S rRNA sequences from 10 strains. Ninety-eight clinical strains were isolated from clinical patient specimens. A total of 98 strains of bacteria were identified by phenotypic methods; PCR with newly designed primers and universal primers. All purified PCR products were sequenced using both forward and reverse primers on an automated DNA analyzer. In this study, we evaluated the usefulness of the newly designed primers and the universal primers for the detection of bacteria, and both these techniques were compared with phenotypic methods for bacteria detection. When we also tested 98 strains of clinical isolates with newly designed primers, about 778 bp DNA fragments were amplified and identified from all strains. Of the 98 strains, 94 strains (95.9%) correspond in comparison with phenotypic methods. The newly designed primers showed that the identities of 98 (100%) strains were the same as those obtained by universal PCR primers. The overall agreement between the newly designed primers and universal primers was 100%. The primer set was designed for rapid, accurate, and cheap identification of bacterial pathogens. We think the newly designed primer set is useful for the identification of pathogenic bacteria.
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문제 정의
The purpose of this study was to evaluate the utility of newly designed 16S rRNA primers in a clinical microbiology laboratory by comparing it with identification by universal PCR primers and the phenotypic methods.
제안 방법
In this study, we evaluated the usefulness of the newly designed primers and of universal primers (8F and 1492R) for identification of bacteria, and both these techniques were compared with phenotypic methods (Vitek, biochemical test, and streptex) for bacteria identification. Ninety-eight clinical strains (50 gram-negative and 48 gram-positive) were isolated from clinical patient specimens.
However, as yet, the usefulness of 16S rRNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rRNA sequencing as a means to identify a collection of isolates obtained from clinical sources.
In this study, we evaluated the utility of newly designed primer as a means to identify 98 isolates obtained from clinical sources. Surveys looked at the feasibility of identifying medically important bacteria using newly designed primers.
In this study, we evaluated the utility of newly designed primer as a means to identify 98 isolates obtained from clinical sources. Surveys looked at the feasibility of identifying medically important bacteria using newly designed primers. When we also tested 98 strains of clinical isolates with newly designed primers and universal primers, DNA fragment was amplified and was identified from all strains.
Phenotypic methods for identification of strains appear to be unreliable. The purpose of the present study was to compare commonly used phenotypic methods, the Vitek system and PCR amplicon-sequencing based methods targeting the 16S rRNA gene.
In other words, twenty of 20 strains were identified as the same species by newly designed primers, in agreement with the naming by the ATCC or NCCP. Then, we evaluated the usefulness of this primer in the identification of isolates clinically significant bacterial strains that showed phenotypic identification methods. A total of 98 bacterial strains were tested by molecular method including the newly designed primers with universal primers and phenotypic methods.
대상 데이터
For the specificity of conventional PCR assay with designed primer, it was verified using strains of both American Type Culture Collection (ATCC; Manassas, VA, USA) reference and clinical origins. 20 strains obtained from the ATCC, were used in this study. And 98 clinical (50 gram-negative and 48 gram-positive) strains were isolated from patient specimens.
This pairs were used to 16S rRNA amplification from the 20 species type strains (Acinetobacter lwoffii ATCC 17925, Bacillus subtilis ATCC 6633, Brevibacilus brevis ATCC 8246, Enterobacter cloacae ATCC 700323, Enterobacter sakazakii ATCC 29544, Enterococcus casseliflavus ATCC 700327, Enterococcus faecalis ATCC 51299, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Escherichia coli O157:H7 ATCC 43888, Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 7005, Providencia alcalifaciens ATCC 51902, Pseudomonas aeruginosa ATCC 9027, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 1925, Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619, Streptococcus pyogenes ATCC 19615, Vibrio parahaemolyticus NCCP 10511), all of which were amplified successfully and showed no additional bands. The newly designed primers showed about 778 bp size DNA band (PCR product) in the 98 clinical isolated strains.
성능/효과
72% (Table 2). All strains, except Bacillus subtilis, analysed sequences revealed identification above 99% (99.2~100%), Bacillus subtilis is identified as having a high rate of 98.9%. The identities of 19 type strains were the same as those obtained by universal primers and the rate of identification is above 99%.
These misidentifications did lead to errors in interpretation of clinical isolates. However, in this study, Newly designed PCR primers and universal PCR primers for K. pneumoniae, Serratia marcescens, Morganella morganii ssp morganii, Proteus mirabilis, Citrobacter braakii and Acinetobactor baumanii complex showed good agreement with phenotypic methods. With the exception of 3 isolates (S.
After all, identification of phenotypic method of 4 isolates was not discriminative enough. However, new primers and universal primers of these 98 isolates were compared to the known 16S rRNA gene sequences in the GenBank, yielded the correct identity, with good discrimination. The overall agreement between the newly designed primers and universal primers were 100%.
The overall agreement between the newly designed primers and universal primers were 100%. Newly designed PCR primers and universal PCR primers analysis of the clinical isolates revealed same results for 50 gram negative isolates, while phenotypic result of 1 isolate was inconsistent. One Citrobacter freundii strain was incorrectly identified as E.
Four of 98 isolated strains showed a considerable number of mismatches with clinical isolates in phenotypic method. Newly designed PCR primers and universal PCR primers analysis of the isolates revealed same results for 98 clinical isolates, while in 4 isolates, phenotypic method were inconsistent (Table 3). It reaffirms the 16S rRNA genes amplification of the 4 isolates showed bands at about 1,492 bp and 778 bp, respectively.
An observed percentage difference is the number of base mismatches between two aligned sequences, as determined. Obtained full sequences showed approximately average 99.74% (99.4~99.8%) identification with the database sequences using universal primers, indicating that correct amplification had been achieved. Also, average bacteria identification by new primers were 99.
) were studied to assess and compare newly designed PCR primers and universal PCR primers by 16S rRNA-based identification of bacteria. The 4 strains (E. coli, S. aureus, C. striatum, Streptococcus sp.) has been identified as a strain of Citrobacter freundii, Stapylococcus saprophyticus, Corynebacterium confusum, Arthrobacter cumminsii, respectively (Table 3). After all, identification of phenotypic method of 4 isolates was not discriminative enough.
9%. The identities of 19 type strains were the same as those obtained by universal primers and the rate of identification is above 99%. For all 20 type strains, there was <1.
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