Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR

BACKGROUND: Broad-range PCR provides valuable information for detecting bacterial infections. This study assesses the combined use of broad-range real-time PCR and high-resolution melting analysis for rapid detection and identification of clinically important bacteria. METHODS: We subjected 46 bacterial culture colonies representing 25 clinically important bacterial species to LightCycler real-time PCR amplification of the 16S rRNA gene in the presence of LCGreen I fluorescent dye. We performed high-resolution melting analysis of the PCR products with the HR-1 instrument and used melting profiles as molecular fingerprints for bacterial species identification. We validated this method via assessment of 54 consecutive bacteria culture colonies obtained from a clinical microbiology laboratory. RESULTS: The 16S rRNA gene of all 25 bacterial species was amplifiable by this method, with PCR product lengths of 216 or 217 bp. Of the 25 bacterial species, we identified 11 via a 1-step post-PCR high-resolution melting analysis. The remaining bacterial species were identified via the high-resolution melting plots obtained by heteroduplex formation between the PCR products of the tested and reference bacterial species or by a 2nd real-time PCR targeting a different region of the 16S rRNA gene. A high-resolution melting database and a working protocol were established for identifying these 25 bacterial species. In the validation assay, a 94% accuracy rate was achieved when the bacterial species were in the high-resolution melting database. CONCLUSIONS: This assay requires no multiplexing or hybridization probes and provides a new approach for bacterial species identification in a molecular diagnostic laboratory.     

Distribution and antimicrobial resistance of bloodstream pathogens in surgical and medical ICUs北大核心CSCD

Objective To analyze the distribution and antimicrobial resistance of bloodstream pathogens in surgical intensive care unit (SICU) and medical intensive care unit (MICU) of Fujian Provincial Hospital in the past four and half years for better use of antimicrobial drugs. Methods A retrospective analysis was carried out for the bloodstream pathogens isolated from SICU and MICU patients from January 2012 to June 2016. The clinical data and outcomes of patients were also reviewed. Results A total of 329 strains of isolates were recovered from blood samples in SICU, including gram-negative bacteria (53.5%), gram-positive bacteria (39.2%), and fungi (7.3%); 258 strains were collected from MICU, including gram-negative bacteria (57.8%), gram-positive bacteria (36.0%), and fungi (6.2%). A. baumannii, K. pneumonia and E. coli were the top three gram-negative species in both SICU and MICU. The main gram-positive species were coagulasenegative Staphylococcus and Enterococcus faecium. Overall, 386 cases of bloodstream infections were diagnosed, including 226 cases in SICU (202 cases of single bacterial infection and 24 cases of multiple bacterial infection), and 160 cases in MICU (138 cases of single bacterial infection and 22 cases of multiple bacterial infection). A. baumannii isolates showed significantly higher rate of resistance to antibiotics in SICU than in MICU, while the K. pneumoniae and E. coli isolates in MICU showed higher resistance rates to cephalosporins, quinolones, penicillins and carbapenems than the corresponding isolates in SICU. The coagulase negative Staphylococcus and E. faecium isolates in MICU were associated with significantly higher resistance rates to quinolones and tigecycline than those strain in SICU. The bloodstream infections due to K. pneumoniae, E. coli and E. faecium were associated with higher mortality in MICU than in SICU, while the bloodstream infections due to A. baumannii were associated with higher mortality in SICU than in MICU. The total mortality rate of bloodstream infections was higher in MICU than in SICU. Conclusions SICU and MICU share similar profile of main bloodstream pathogens even though the disease spectrum was different between SICU and MICU. All the bloodstream pathogens isolated from MICU patients except A. baumannii showed significantly higher antimicrobial resistance rates than the isolates from SICU. The mortality rate associated with bloodstream infection was also higher in MICU patients than in SICU. © 2018, Editorial Department of Chinese Journal of Infection. All rights reserved.     

分子诊断学在细菌和真菌的血流感染检测中的应用

血流感染（bloodstream infection,BSI）是严重的感染性疾病.血液培养被认为是诊断血液感染的金标准,但是要花费数日鉴定出病原体.能够快速、准确地鉴定血流感染的病原体,可以明显改善病人预后.随着技术进步,分子诊断与临床实验室诊断关系愈加密切,能够为血流感染提供快速、准确的诊断.文章对分子诊断学在细菌和真菌性血流感染检测中的作用进行综述.     

Routine use of a real-time polymerase chain reaction method for detection of bloodstream infections in neutropaenic patients

We examined the performance of a real-time polymerase chain reaction (PCR) test (SeptiFast) for early detection of bloodstream infection in febrile neutropaenic patients. Blood samples from 201 patients were screened for pathogens by blood culture and by PCR on the first day of fever. PCR results were available earlier (median 3 days for bacteria, 5 days fungal pathogens; P ≤ 0.01). The sensitivity (0.74) and specificity (0.96) of the PCR test were acceptable for Gram negatives when culture was considered the gold standard, but sensitivity of the test was poorer for Gram-positive organisms (0.39). The PCR assay also led to 22.9% of invalid results. SeptiFast speeds the microbiological diagnosis of bloodstream infection in neutropaenic patients. However, the frequent failure of instrumental control procedures, the relatively poor sensitivity of the test, and the lack of phenotypic data on antimicrobial susceptibility associated with its high costs suggest that this assay cannot replace the blood cultures.     

头孢哌酮-舒巴坦对非发酵革兰阴性杆菌的抗菌作用

目的 :评价头孢哌酮 舒巴坦 (CPZ SBT)对非发酵革兰阴性杆菌临床分离株的体外抗菌作用 ,并与其他抗菌药比较。方法 :收集临床分离的非发酵革兰阴性杆菌 ,采用琼脂扩散法进行药敏试验 ,按美国国家临床实验室标准委员会 (NCCLS)2 0 0 0判断结果。结果 :测定 390 5株非发酵革兰阴性杆菌对 19种抗菌药的敏感性。CPZ SBT对铜绿假单胞菌的抗菌作用仅次于亚胺培南和美罗培南 ,与头孢他啶相仿。CPZ SBT对不动杆菌属、产碱杆菌属、伯克霍德尔菌属、嗜麦芽窄食单胞菌和黄杆菌属等均具良好的抗菌作用 ,尤其后 2种菌中多数菌株对碳青霉烯类耐药而对本品则多数敏感。在 390 5株非发酵菌中对CPZ敏感株占 39.5 % ,而对CPZ SBT敏感株增至 70 .4 % ,耐药株则从 37.0 %下降至 10 .8%。结论 :临床分离菌中非发酵革兰阴性杆菌日益增多 ,CPZ SBT在非发酵革兰阴性杆菌感染中可能有良好的应用前景。     

Usefulness of multilocus polymerase chain reaction followed by electrospray ionization mass spectrometry to identify a diverse panel of bacterial isolates

Polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was tested for its ability to accurately identify a blinded panel of 156 diverse bacterial isolates, mostly human and/or animal pathogens. Here, 142/156 (91%) isolates were correctly identified to the genus level and 115/156 (74%) were correctly identified to the species level. Only 9% were misidentified. This study shows that multilocus PCR/ESI-MS has the potential to be a useful technique for identifying a broad range of bacteria.     

A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens

Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens.     

Healthcare-associated bloodstream infection: A distinct entity? Insights from a large U.S. database

OBJECTIVE: To gain a better understanding of the epidemiology, microbiology, and outcomes of early-onset, culture-positive, community-acquired, healthcare-associated, and hospital-acquired bloodstream infections. DESIGN: We analyzed a large U.S. database (Cardinal Health, MediQual, formerly MedisGroups) to identify patients with bacterial or fungal bloodstream isolates from 2002 to 2003. SETTING: The data set included administrative and clinical variables (physiologic, laboratory, culture, and other clinical) from 59 hospitals. Bloodstream infections were identified in those hospitals collecting clinical and culture data for at least the first 5 days of admission. PATIENTS: Patients with bloodstream infection within 2 days of admission were classified as having community-acquired bloodstream infection. Those with a prior hospitalization within 30 days, transfer from another facility, ongoing chemotherapy, or long-term hemodialysis were classified as having healthcare-associated bloodstream infection. Bloodstream infections that developed after day 2 of admission were classified as hospital-acquired bloodstream infection. A total of 6,697 patients were identified as having bloodstream infection. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Healthcare-associated bloodstream infection accounted for more than half (55.3%) of all bloodstream infections. Nearly two thirds (62.3%) of hospitalized patients with bloodstream infection suffered from either hospital-acquired bloodstream infection or healthcare-associated bloodstream infection and had higher morbidity and mortality rates than those with community-acquired bloodstream infection. Of all bloodstream infection pathogens, fungal organisms were associated with the highest crude mortality, longest length of stay in hospital, and greatest total charges. Of all bacterial bloodstream infections, methicillin-resistant Staphylococcus aureus was associated with the highest crude mortality rate (22.5%), the longest mean length of stay (11.1 +/- 10.7 days), and the highest median total charges ($36,109). After we controlled for confounding factors, methicillin-resistant S. aureus was associated with the highest independent mortality risk (odds ratio 2.70; confidence interval 2.03-3.58). S. aureus was the most commonly encountered pathogen in all types of early-onset bacteremia. CONCLUSIONS: Healthcare-associated bloodstream infection constitutes a distinct entity of bloodstream infection with its unique epidemiology, microbiology, and outcomes. Methicillin-resistant Staphylococcus aureus carries the highest relative mortality risk among all pathogens.     

Shigellosis with resultant septic shock and renal failure

Septic shock is a rare, potentially life-threatening complication of bacterial dysentery. The clinical presentation of septic shock includes hypotension, bleeding, hypoxia, acidosis, and jaundice. Historically gram-negative organisms were the most frequent cause of nosocomial bloodstream infections. However isolation of gram-positive organisms has become increasingly frequent with Staphylococcus species accounting for over one half of all nosocomial bloodstream pathogens. Bacterial dysentery is an acute diarrheal illness characterized by abdominal cramping, fever, and the production of mucoid, bloody stools. Laboratory findings include positive stool culture and increased leukocytes in direct fecal exam. Chemistry and hematology values may be abnormal. The disease is usually self-limiting but administration of antibiotics and rehydration therapy may be warranted in severe cases. This case study describes a 53 year old male who presented with diarrhea and diabetic acidosis and subsequently developed respiratory distress and renal failure due to shigellosis. Discussion of disease pathogenesis and treatment are provided.     

细菌革兰双检荧光定量PCR方法检测新生儿败血症

目的 建立细菌革兰阴、阳性菌双重实时荧光定量检测体系,探讨其检测败血症的临床应用价值.方法 分析细菌16SrRNA基因序列,在高度保守区自行设计通用引物和革兰阴性和阳性分型探针,选取临床较常见的35株菌株进行细菌革兰双检实时荧光定量PCR方法检测;对临床疑为败血症的512例新生患儿分别做细菌革兰双检荧光定量PCR和血培养检测.结果 细菌革兰双检荧光定量PCR具有较好的敏感性和特异性,能稳定检测到10 CFU左右细菌数.35株菌株进行细菌革兰双检荧光定量PCR检测,均为阳性,且革兰阴、阳性菌能正确分型和定量.巨细胞病毒、EB病毒、乙肝病毒、新型隐球菌及白色念珠菌、人基因组DNA及空白对照均为阴性.对临床疑为败血症的512份新生患儿标本中,革兰双检荧光定量PCR检测血标本阳性率8.20%(42/512),血培养阳性率6.25%(32/512),前者明显高于后者,差异具有统计学意义(χ<'2>=8.10,P<0.01),30例非感染性疾病同期患儿血标本革兰双检荧光定量PER及细菌培养均为阴性.若以血培养作为对照,细菌革兰双检荧光定量PCR方法的诊断敏感度为100%,特异度为97.92%,准确性98.05%.结论 建立了用通用引物和分型双荧光探针的细菌革兰双检荧光定量PER方法.其检测快速、准确,具有很大的临床推广价值.     

High-throughput screening of bacterial pathogens in clinical specimens using 16S rDNA qPCR and fragment analysis

Molecular-based detection of bacterial pathogens directly from clinical specimens permits rapid initiation of effective antimicrobial treatment and adequate patient management. Broad-range polymerase chain reaction (PCR) amplification of the 16S rRNA gene (16S rDNA qPCR) is used in many diagnostic laboratories as a complement to cultural identification of bacterial pathogens. However, efforts for automation of 16S rDNA PCR workflows are needed in order to reduce turnaround times and to enhance reproducibility and standardization of the technique. In this retrospective method evaluation study, clinical specimens (N?=?499) from patients with suspected bacterial infections were used to evaluate 2 diagnostic semiautomated workflows for rapid bacterial pathogen detection. The workflows included automated DNA extraction (QIASymphony), 16S rDNA qPCR, fragment or melting curve analysis, and amplicon sequencing. Our results support the use of the 16S rDNA qPCR and fragment analysis workflow as it enabled rapid and accurate identification of bacterial pathogens in clinical specimens.     

肝移植术后细菌感染的常见病原菌及其耐药性

目的　探讨肝移植术后细菌感染的常见病原菌及其耐药性 ,为肝移植术后细菌感染的经验性防治提供用药依据。方法　对 2 4例肝移植、肝肾联合移植患者 1个月内胆汁、引流液、咽拭子等标本培养阳性的细菌种类、耐药性及其发生时间进行分析。结果　肝移植术后细菌感染以金黄色葡萄球菌、阴沟杆菌、溶血性葡萄球菌、屎肠球菌最常见 ,对临床常用的抗生素耐药性高 ,革兰阳性菌对万古霉素敏感性高 ,革兰阴性菌对替考拉宁和泰能敏感性高。结论　肝移植早期积极的细菌培养对临床抗生素的应用有指导意义。肝移植术后细菌感染以金黄色葡萄球菌、阴沟杆菌、溶血性葡萄球菌、屎肠球菌最常见 ,临床可疑细菌感染而尚未获得培养结果可经验性加用泰能或万古霉素、替考拉宁。     

Nosocomial infections in medical intensive care units in the United States. National Nosocomial Infections Surveillance System.

OBJECTIVE: To describe the epidemiology of nosocomial infections in medical intensive care units (ICUs) in the United States. DESIGN: Analysis of ICU surveillance data collected through the National Nosocomial Infections Surveillance (NNIS) System between 1992 and 1997. SETTING: Medical ICUs in the United States. PATIENTS: A total of 181,993 patients. MEASUREMENTS AND MAIN RESULTS: Nosocomial infections were analyzed by infection site and pathogen distribution. Urinary tract infections were most frequent (31%), followed by pneumonia (27%) and primary bloodstream infections (19%). Eighty-seven percent of primary bloodstream infections were associated with central lines, 86% of nosocomial pneumonia was associated with mechanical ventilation, and 95% of urinary tract infections were associated with urinary catheters. Coagulase-negative staphylococci (36%) were the most common bloodstream infection isolates, followed by enterococci (16%) and Staphylococcus aureus (13%). Twelve percent of bloodstream isolates were fungi. The most frequent isolates from pneumonia were Gram-negative aerobic organisms (64%). Pseudomonas aeruginosa (21%) was the most frequently isolated of these. S. aureus (20%) was isolated with similar frequency. Candida albicans was the most common single pathogen isolated from urine and made up just over half of the fungal isolates. Fungal urinary infections were associated with asymptomatic funguria rather than symptomatic urinary tract infections (p < .0001). Certain pathogens were associated with device use: coagulase-negative staphylococci with central lines, P. aeruginosa and Acinetobacter species with ventilators, and fungal infections with urinary catheters. Patient nosocomial infection rates for the major sites correlated strongly with device use. Device exposure was controlled for by calculating device-associated infection rates for bloodstream infections, pneumonia, and urinary tract infections by dividing the number of device-associated infections by the number of days of device use. There was no association between these device-associated infection rates and number of hospital beds, number of ICU beds, or length of stay. There is a considerable variation within the distribution of each of these infection rates. CONCLUSIONS: The distribution of sites of infection in medical ICUs differed from that previously reported in NNIS ICU surveillance studies, largely as a result of anticipated low rates of surgical site infections. Primary bloodstream infections, pneumonia, and urinary tract infections associated with invasive devices made up the great majority of nosocomial infections. Coagulase-negative staphylococci were more frequently associated with primary bloodstream infections than reported from NNIS ICUs of all types in the 1980s, and enterococci were a more frequent isolate from bloodstream infections than S. aureus. Fungal urinary tract infections, often asymptomatic and associated with catheter use, were considerably more frequent than previously reported. Invasive device-associated infections were associated with specific pathogens. Although device-associated site-specific infection rates are currently our most useful rates for performing comparisons between ICUs, the considerable variation in these rates between ICUs indicates the need for further risk adjustment.     

Antibacterial activity of the fourth-generation fluoroquinolones gatifloxacin and moxifloxacin against ocular pathogens

The ideal ophthalmic anti-infective exhibits broad-spectrum activity against gram-positive, gram-negative, and atypical bacterial species. These pathogens can cause potentially blinding infections such as keratitis and endophthalmitis, both of which are associated with ophthalmic surgery or traumatic injury. These infections often require aggressive antibacterial therapy, preferably with newer generations of antibiotics. In this study, minimal inhibitory concentration (MIC) values for gatifloxacin and moxifloxacin were determined in vitro against bacterial strains that were isolated from suspected cases of bacterial keratitis and endophthalmitis. The ocular isolates included 7 gram-positive, 4 gram-negative, and 3 atypical bacterial species. Gatifloxacin and moxifloxacin exhibited similar activity against 6 gram-positive organisms: Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Bacillus cereus, and Enterococcus faecalis. MIC90 values for the drugs against these isolates ranged from 0.08 mg/mL to 0.57 mg/mL and were comparable to previously published values against isolates from patients with systemic infections. The MIC90 for gatifloxacin against Streptococcus viridans was 0.22 mg/mL compared with 0.73 mg/mL for moxifloxacin (P = .011). Among the gram-negative isolates, the mean MIC90 for gatifloxacin against Pseudomonas aeruginosa was 1.28 mg/mL compared with 2.60 mg/ mL for moxifloxacin (P = .023). MIC90 values for gatifloxacin against Klebsiella pneumoniae and Enterobacter aerogenes were one fourth to one fifth the values for moxifloxacin. For the atypicals, the MIC90 values for gatifloxacin against Nocardia asteroides and Mycobacterium chelonae were one fourth the corresponding values for moxifloxacin. Gatifloxacin demonstrated a broad spectrum of activity against several key ocular pathogens tested in this study and was at least as effective as moxifloxacin against these pathogens.     

Rapid Polymerase Chain Reaction-based Screening Assay for Bacterial Biothreat Agents

Objectives:  To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens.
Methods:  The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria.
Results:  The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus .
Conclusions:  A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.     

BIOFIRE® Blood Culture IDentification 2 (BCID2) panel for early adaptation of antimicrobial therapy in adult patients with bloodstream infections: a real-life experience

Our objective was to assess the effectiveness of a multiplex PCR panel for blood culture identification (BCID2) on the implementation of appropriate antimicrobial therapy. We conducted a monocentric pre/post study comparing the time to result from direct microscopic examination (DE) to bacterial identification (BI) in positive blood cultures between 2 different periods: P1 without BCID2 and P2 with BCID2. Appropriate treatments prescribed before DE and after DE / BCID2 and after BI / BCID2 were compared using direct proportion comparison and survival analysis. For mono-microbial bloodstream infections, the proportion of appropriate antimicrobial treatment after DE was 50% in P1 vs. 87.5% after BCID2 in P2 (P < 0.001) for Gram-negative bacteria and 33.0% in P1 vs. 64.4% in P2 (P < 0.01) for Gram-positive bacteria. A significant difference (P = 0.04) was recorded with survival curves for Gram positive bacteria. BCID2 seems effective in reducing the time for prescribing appropriate antimicrobials.     

Development of real-time polymerase chain reaction assay for specific detection of Tsukamurella by targeting the 16S rRNA gene

Recently, members of the genus Tsukamurella have been implicated as important etiologic pathogens contributing to bloodstream and pulmonary infections in immunocompromised patients. Tsukamurella species share many features with other mycolic acid-containing genera of the order Actinomycetales and might therefore be misidentified as belonging to one of these genera. We developed a TaqMan-based real-time polymerase chain reaction assay for the rapid and specific detection of infections due to Tsukamurella species. The assay amplifies and detects a 157-bp segment of the 16S rRNA gene of Tsukamurella. The specificity of the assay was confirmed using a panel of DNAs from 12 Tsukamurella strains and 11 strains belonging to 8 phylogenetic closely related genera. The sensitive and specific nature of the assay provides a valuable tool for the early and precise diagnosis of Tsukamurella infections in clinical diagnostic laboratories.     

Multiplex real-time SYBR Green I PCR assay for detection of tetracycline efflux genes of Gram-negative bacteria

In an effort to find a rapid, efficient, and reliable method for screening and classifying large numbers of tetracycline-resistant bacterial isolates, we developed a multiplex, real-time PCR assay using SYBR Green I and the Roche LightCycler. The assay can rapidly identify eight genes encoding tetracycline resistance efflux pumps including tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H) and tet(J). Primers were selected for PCR amplification of these eight tetracycline resistance determinant (tet) genes commonly found in Gram-negative organisms. We combined primer pairs together to make a single-tube multiplex PCR reaction followed by melting curve analysis. Amplification of the expected tet gene products was confirmed by both agarose gel electrophoresis and DNA sequence analysis. Based on melting temperature differences, we could identify the different classes of tet genes. To test the multiplex PCR, the assay was used on 107 tetracycline-resistant clinical isolates of various Gram-negative organisms isolated in several locations around the world. About 49.5% of those strains carried a tet(A) gene, 35.5% carried a tet(B), 7.5% carried a tet(J), 5.6% carried a tet(C) and 1.9% carried a tet(D) gene. DNA sequence analysis of the amplicons confirmed that the specificity of the test was 100%. The sensitivity of the multiplex test varied from 10 to 1000 CFU per PCR reaction. Our real time PCR assay utilizing SYBR Green I and melting point analysis on the Lightcycler system showed not only a high confidence level in differentiation of the classes of tet genes but also precise reproducibility. Our multiplex PCR tet gene class identification assay offers a significant savings of time and labor in the analysis of large numbers of clinical strains compared with assays using individual gene PCR or traditional phenotype methods.     

Direct identification of chlamydiae from clinical samples using a DNA microarray assay: a validation study

While DNA microarrays have become a widely accepted tool for mRNA expression monitoring, their use in rapid diagnosis of bacterial and viral pathogens is only emerging. So far, insufficient sensitivity and high costs have been the major limiting factors preventing more widespread use of microarray platforms in direct testing of clinical samples. In the present study, a total of 339 samples, among them 293 clinical specimens from animals and humans, were examined by the ArrayTube (AT) DNA microarray assay to detect chlamydial DNA and identify the species of Chlamydia and Chlamydophila involved. Samples included nasal and conjunctival swabs, formalin-fixed, paraffin-embedded and fresh organ tissue, milk, feces and cell culture. Notably, the AT test was shown to detect mixed infections in clinical samples. The calculated median sensitivity of 0.81 over the entire panel of clinical samples was comparable to conventional 16S PCR, but slightly lower than real-time PCR and other PCR assays. However, when a panel of long-time stored swab samples was excluded from the calculation, the sensitivity was clearly higher (0.87) and equivalent to that of real-time PCR. Altogether, the data demonstrate the suitability of this DNA microarray assay for routine diagnosis.     

23S rRNA基因在临床常见致病菌检测中的应用

目的　根据临床常见致病菌 2 3SrRNA基因序列的差异 ,建立可初步鉴别革兰阴性菌和革兰阳性菌的分子生物学方法。方法　分析常见细菌的 2 3SrRNA基因序列 ,设计相应引物。采用多重PCR扩增标准菌株及临床分离株 2 3SrRNA基因 ,并根据电泳结果作出初步分类。结果　革兰阴性菌株均出现 1条DNA电泳条带(约为 35 0bp) ,而革兰阳性菌株则出现 2条电泳条带 (约为 2 5 0和 4 0 0bp)。 6 0株临床分离菌经PCR扩增、电泳后 ,电泳分析结果与常规鉴定结果符合率达 10 0 %。结论　 2 3SrRNA基因检测用于细菌初步分类鉴定 ,具有快速、灵敏、准确的特点 ,可为细菌感染的实验室诊断提供客观依据