基因芯片技术检测鉴定临床常见致病真菌的初步研究

目的为了快速、简便、高通量地鉴定临床常见致病真菌,建立了一种采用基因芯片技术对临床常见的致病真菌鉴定的分子生物学方法。方法以5.8S rDNA与28S rDNA间的内转录间区2(internal transcribed spacer-2,ITS-2)为靶标,针对待检的临床常见致病真菌设计合成一系列寡核苷酸探针,制成寡核苷酸芯片。待检真菌DNA经通用引物扩增标记后,与芯片杂交,对杂交图谱分析归纳,得到一套种特异性的典型杂交图谱。待检的样品菌与基因芯片杂交,得到的杂交结果与典型图谱比对即可判断出样品的种类。结果以涉及8个属20个种的标准致病真菌菌株对芯片的特异性、重复性、灵敏度进行考察,结果表明,该研究建立的基因芯片技术可以有效地区分20种临床常见致病真菌,特异性良好,重复性良好(信噪比CV<10%),灵敏度为15 pg/ml真菌DNA。收集从临床分离的84株致病真菌菌株对基因芯片进行试用,结果显示基因芯片的鉴定结果与常规鉴定方法的鉴定结果一致。结论这项技术的建立可以稳定、特异性地实现临床常见致病真菌的高通量鉴定,为进一步检测研究奠定了基础。     

An oligonucleotide microarray for the detection of vaccinia virus

Vaccinia virus is a member of the orthopoxvirus group, to which also belongs variola virus, one of the most hazardous pathogens known to man. To establish a model system to detect orthopoxviruses, a vaccinia oligonucleotide microarray is designed, produced and tested. Vaccinia virus is used to test the prepared microarrays. The virus DNA samples in different propagation phases are extracted and hybridised with the oligonucleotide microarray. The results showed that the oligonucleotide microarray can detect vaccinia virus with high specificity and sensitivity.     

Simultaneous analysis of foodborne pathogenic bacteria by an oligonucleotide microarray assay

A rapid and accurate method for simultaneous identification of foodborne infectious pathogens was developed based on oligonucleotide microarray technology. The proposed identification method is based on PCR amplification of the target region of the groEL genes with degenerate primers, followed by the PCR products hybridization with oligonucleotide probes specific for species. The groEL gene amplification products of seventeen species of pathogenic bacteria were hybridized to the oligonucleotide array. Hybridization results were analyzed with digoxigenin-linked enzyme reaction. Results indicated that fifteen species of pathogenic bacteria showed high sensitivity and specificity for the oligonucleotide array, while two other species gave cross-reaction with the E. coli. Our results suggested that microarray analysis of foodborne infectious pathogens might be very useful for simultaneous identification of bacterial pathogens. The oligonucleotide array can also be applied to samples collected in clinical settings of foodborne infections. The superiority of oligonucleotide array over other tests lies on its rapidity, accuracy and efficiency in the diagnosis, treatment and control of foodborne infections.     

Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray

We coupled multiplex PCR and a DNA microarray to construct an assay suitable for the simultaneous detection of five important marine fish pathogens (Vibrio vulnificus, Listonella anguillarum, Photobacterium damselae subsp. damselae, Aeromonas salmonicida subsp. salmonicida, and Vibrio parahaemolyticus). The array was composed of nine short oligonucleotide probes (25-mer) complementary to seven chromosomal loci (cyt, rpoN, gyrB, toxR, ureC, dly, and vapA) and two plasmid-borne loci (fatA and A.sal). Nine primer sets were designed to amplify short fragments of these loci (100 to 177 bp) in a multiplex PCR. PCR products were subsequently labeled by nick translation and hybridized to the microarray. All strains of the five target species (n = 1 to 21) hybridized to at least one species-specific probe. Assay sensitivities ranged from 100% for seven probes to 83 and 67% for the two remaining probes. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria (n = 40 strains; 100% specificity). Using purified genomic DNA, we were able to detect PCR products with < 20 fg of genomic DNA per reaction (equivalent to four or five cells), and the array was at least fourfold more sensitive than agarose gel electrophoresis for detecting PCR products. In addition, our method allowed the tentative identification of virulent strains of L. anguillarum serotype O1 based on the presence of the fatA gene (67% sensitivity and 100% specificity). This assay is a sensitive and specific tool for the simultaneous detection of multiple pathogenic bacteria that cause disease in fish and humans.     

Simultaneous detection and identification of Candida, Aspergillus, and Cryptococcus species by reverse line blot hybridization

We report on a reverse line blot (RLB) assay, utilizing fungal species-specific oligonucleotide probes to hybridize with internal transcribed spacer 2 region sequences amplified using a nested panfungal PCR. Reference and clinical strains of 16 Candida species (116 strains), Cryptococcus neoformans (five strains of Cryptococcus neoformans var. neoformans, five strains of Cryptococcus neoformans var. grubii, and six strains of Cryptococcus gatti), and five Aspergillus species (68 strains) were all correctly identified by the RLB assay. Additional fungal species (16 species and 26 strains) not represented on the assay did not exhibit cross-hybridization with the oligonucleotide probes. In simulated clinical specimens, the sensitivity of the assay for Candida spp. and Aspergillus spp. was 10(0.5) cells/ml and 10(2) conidia/ml, respectively. This assay allows sensitive and specific simultaneous detection and identification of a broad range of fungal pathogens.     

Identification of medically important molds by an oligonucleotide array

Infections caused by fungi have increased in recent years. Accurate and rapid identification of fungal pathogens is important for appropriate treatment with antifungal agents. On the basis of the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 64 species (32 genera) of clinically important filamentous (or dimorphic) fungi. These 64 species included fungi causing superficial, cutaneous, subcutaneous, and invasive infections. The method consisted of PCR amplification of the ITS regions using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species- or group-specific oligonucleotides immobilized on a nylon membrane. Of 397 fungal strains (290 target and 107 nontarget strains) tested, the sensitivity and specificity of the array was 98.3% (285/290) and 98.1% (105/107), respectively. Misidentified strains were usually those belonging to the same genus of the target species or having partial homology with oligonucleotide probes on the membrane. The whole procedure can be finished within 24 h starting from isolated colonies; reproductive structures, which are essential for the conventional identification methods, are not needed. In conclusion, the present array is a powerful tool for identification of clinically important filamentous fungi and may have the potential to be continually extended by adding further oligonucleotides to the array without significantly increasing the cost or complexity.     

Simple PCR-Based DNA Microarray System To Identify Human Pathogenic Fungi in Skin

Fungal diseases in immunocompromised hosts pose significant threats to their prognoses. An accurate diagnosis and identification of the fungal pathogens causing the infection are critical to determine the proper therapeutic interventions, but these are often not achieved, due to difficulties with isolation and morphological identification. In an effort to ultimately carry out the simultaneous detection of all human pathogenic microbes, we developed a simple system to identify 26 clinically important fungi by using a combination of PCR amplification and DNA microarray assay (designated PCR-DM), in which PCR-amplified DNA from the internal transcribed spacer region of the rRNA gene was hybridized to a DNA microarray fabricated with species-specific probes sets using the Bubble Jet technology. PCR-DM reliably identified all 26 reference strains; hence, we applied it to cases of onychomycosis, taking advantage of the accessibility of tissue from skin. PCR-DM detected fungal DNA and identified pathogens in 92% of 106 microscopy-confirmed onychomycosis specimens. In contrast, culture was successful for only 36 specimens (34%), 3 of which had results inconsistent with the results of PCR-DM, but sequence analysis of the isolates proved that the PCR-DM result was correct. Thus, PCR-DM provides a powerful method to identify pathogenic fungi with high sensitivity and speed directly from tissue specimens, and this concept could be applied to other fungal or nonfungal infectious human diseases in less accessible anatomical sites.The identification of a given pathogen is a fundamental step for appropriate treatment of infectious diseases. Isolating such pathogens by culture is generally performed to confirm the diagnoses of fungal diseases, but it is time-consuming and fungal outgrowth can take weeks. Among the common fungal diseases, candidiasis and aspergillosis can cause serious diseases in patients receiving immunosuppressive chemotherapy for cancer or suffering from immunodeficiency because of HIV infection. These invasive infections are often associated with poor prognoses, and accurate identification of fungal pathogens at the species level is important for the prompt initiation of antifungal treatments. It is thus desirable that a method which can systematically identify pathogens directly from patient specimens be developed. Taking advantage of the accessibility of tissue from skin, we selected fungal infection in the nail, onychomycosis, as a model for clinical application of the DNA microarray-based technology for the detection of a broad spectrum of species causing human fungal diseases. Most cutaneous fungal infections are superficial; are limited to the stratum corneum, hair, and nails; and are caused by Trichophyton spp. However, a spectrum of pathogens are also identified as causes of systemic infections (7, 14, 16, 23). Onychomycosis is a common disease of the nail caused by dermatophytes, yeasts, and other molds. Direct microscopic examination of nail specimens is generally performed to diagnose onychomycosis. However, attempts to isolate pathogens from nail specimens confirmed to contain fungal elements often yield negative results and fail to provide a species identification (19, 20). For these reasons, onychomycosis could serve as a relevant model to which we may apply our new DNA microarray system.In recent years, numerous DNA-based methods have been developed to diagnose infectious diseases and to identify pathogens. PCR has proven to be useful for the rapid diagnosis of a variety of infectious diseases (3, 6, 22, 26). The rRNA gene cassette, particularly the large-subunit 28S rRNA gene, is well conserved among species and provides useful information for phylogenetic analysis (28). The internal transcribed spacer (ITS) regions of the rRNA gene have also been used for phylogenetic analyses (5, 25, 27) and to detect and identify human fungal pathogens (1, 2, 12, 30). The majority of the currently available methods rely on PCR with species-specific probes and therefore are limited in their multiplexing capabilities, resulting in high costs if all relevant species need to be considered or requiring a longer time if DNA sequences are to be determined. More recently, the DNA microarray assay was developed. It permits the simultaneous detection and analysis of thousands of genes in a short assay time and, recently, with greater cost-efficiency, and it has proven useful for the identification of pathogens in a variety of clinical settings (6, 8, 13).Although the methods used to genetically identify pathogens have been vigorously studied, most of these were done using genomic DNA extracted from fungal isolates that were cultured from infected tissues (8, 29). This approach provides high specificity but does not exceed conventional methods in terms of time. In the present study, we generated a panel of oligonucleotide probes which correspond to unique sequences of the ribosomal ITS regions of fungal pathogens that are commonly encountered in clinical practice. Multiple sets of DNA microarrays were fabricated using the Bubble Jet technology, which ejects picoliter amounts of oligonucleotide probe solutions accurately as a unique matrix onto specially coated glass slides (21). The performance of this simple combination of PCR amplification and DNA microarray assay (PCR-DM) was evaluated for its sensitivity and specificity by using 87 reference fungal strains and 106 infected human nail tissues and comparing the results with those of the conventional culture technique.     

Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections

The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples.     

PCR detection of DNA specific for Aspergillus species in serum of patients with invasive aspergillosis.

We investigated the possible presence of DNA specific for Aspergillus species in serum samples of patients with invasive aspergillosis (IA) by the nested PCR method. Fourteen strains of fungi including 5 strains of Aspergillus species and 10 strains of common bacteria were used for examination of specificity and sensitivity of the nested PCR. Two sets of oligonucleotide primers were derived from the sequence of the variable regions V7 to V9 of the 18S rRNA genes of Aspergillus fumigatus. The specific fragment was amplified from five strains of Aspergillus species in the single and nested PCR but not from other microorganisms. Target DNA was detected by the nested PCR with as little as 50 fg of the extracted DNA of A. fumigatus. We investigated the detection of DNA specific for Aspergillus species in serum samples of a murine model of aspergillosis and 20 patients with IA. The specific fragment was detected by the nested PCR in 71% of serum samples of infected mice and 70% of serum samples of patients with IA, while galactomannan antigen was detected in 43 and 60% of samples, respectively. The high sensitivity and specificity of the nested PCR indicate that the assay can provide early diagnosis with sufficient accuracy to be clinically useful for immunocompromised patients with IA.     

Simultaneous detection of four human pathogenic microsporidian species from clinical samples by oligonucleotide microarray

Microsporidian species have been rapidly emerging as human enteric pathogens in immunocompromised and immunocompetent individuals in recent years. Routine diagnostic techniques for microsporidia in clinical laboratories are laborious and insensitive and tend to underestimate their presence. In most instances, they are unable to differentiate species of spores due to their small sizes and similar morphologies. In this study, we report the development of another protozoan oligonucleotide microarray assay for the simultaneous detection and identification to the species level of four major microsporidian species: Enterocytozoon bieneusi, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis. The 18S small-subunit rRNA gene was chosen as the amplification target, labeled with fluorescence dye, and hybridized to a series of species-specific oligonucleotide probes immobilized on a microchip. The specificity and sensitivity of the microarray were clearly demonstrated by the unique hybridization profiles exhibited by each species of microsporidian tested and its ability to detect as few as 10 spores. In order to assess the applicability of this microarray in a clinical setting, we conducted microarray assays of 20 fecal samples from AIDS patients. Twelve of these samples were positive for the presence of microsporidia and could be confidently identified; 11 of them were positive for more than one species. Our results suggested that this microarray-based approach represents an attractive diagnostic tool for high-throughput detection and identification of microsporidian species in clinical and epidemiological investigations.     

Development of an Oligonucleotide Array for Direct Detection of Fungi in Sputum Samples from Patients with Cystic Fibrosis

Cystic fibrosis (CF) is the most common inherited genetic disease in Caucasian populations. Besides bacteria, many species of fungi may colonize the respiratory tract of these patients, sometimes leading to true respiratory infections. In this study, an oligonucleotide array capable of identifying 20 fungal species was developed to directly detect fungi in the sputum samples of CF patients. Species-specific oligonucleotide probes were designed from the internal transcribed spacer (ITS) regions of the rRNA operon and immobilized on a nylon membrane. The fungal ITS regions were amplified by PCR and hybridized to the array for species identification. The array was validated by testing 182 target strains (strains which we aimed to identify) and 141 nontarget strains (135 species), and a sensitivity of 100% and a specificity of 99.2% were obtained. The validated array was then used for direct detection of fungi in 57 sputum samples from 39 CF patients, and the results were compared to those obtained by culture. For 16 sputum samples, the results obtained by the array corresponded with those obtained by culture. For 33 samples, the array detected more fungal species than culture did, while the reverse was found for eight samples. The accuracy of the array for fungal detection in sputum samples was confirmed (or partially confirmed) in some samples by cloning and resequencing the amplified ITS fragments. The present array is a useful tool for both the simultaneous detection of multiple fungal species present in the sputa of CF patients and the identification of fungi isolated from these patients.     

Detection and identification of fungal pathogens in blood by using molecular probes.

A PCR assay was developed for the detection and identification of Candida and Aspergillus species. The design of the oligonucleotide primer pair as well as the species-specific probes used for species identification was derived from a comparison of the sequences of the 18S rRNA genes of various fungal pathogens. The primers targeted a consensus sequence for a variety of fungal pathogens. The assay was tested for sensitivity and specificity with 134 fungal and 85 nonfungal isolates. To assess clinical applicability, 601 blood samples from four defined groups were tested: group A (n = 35), controls; groups B to D (n = 86), patients with febrile neutropenia, without fungal colonization (group B; n = 29) and with fungal colonization (group C; n = 36); and patients with documented invasive fungal infection (IFI) (group D; n = 21). The assay detected and, by species-specific hybridization, identified most of the clinically relevant Candida and Aspergillus species at 1 CFU/ml of blood. Amplification was 100% sensitive for all molds and yeasts tested, with Histoplasma capsulatum being the only non-Aspergillus species hybridizing with the Aspergillus spp. probe. None of 35 group A patients and only 3 of 65 group B and C patients were PCR positive. The sensitivity of the assay for specimens from patients with IFI (21 patients in group D) was 100% if two specimens were tested. For specificity, 3 of 189 specimens from patients at risk but with negative cultures were positive by the assay, for a specificity of 98%. PCR preceded radiological signs by a median of 4 days (range, 4 to 7 days) for 12 of 17 patients with hepatosplenic candidiasis or pulmonary aspergillosis. For the 10 patients with IFI responding to antifungal therapy, PCR assays became persistently negative after 14 days of treatment, in contrast to the case for 11 patients, who remained PCR positive while not responding to antifungal therapy. Thus, the described PCR assay allows for the highly sensitive and specific detection and identification of fungal pathogens in vitro and in vivo. Preliminary data from the screening of a selected group of patients revealed some value in the early diagnosis and monitoring of antifungal therapy.     

Development of a DNA microarray for detection and identification of fungal pathogens involved in invasive mycoses

Invasive fungal infections have emerged as a major cause of morbidity and mortality in immunocompromised patients. Conventional identification of pathogenic fungi in clinical microbiology laboratories is time-consuming and, therefore, often imperfect for the early initiation of an adequate antifungal therapy. We developed a diagnostic microarray for the rapid and simultaneous identification of the 12 most common pathogenic Candida and Aspergillus species. Oligonucleotide probes were designed by exploiting the sequence variations of the internal transcribed spacer (ITS) regions of the rRNA gene cassette to identify Candida albicans, Candida dubliniensis, Candida krusei, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida lusitaniae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus. By using universal fungal primers (ITS 1 and ITS 4) directed toward conserved regions of the 18S and 28S rRNA genes, respectively, the fungal ITS target regions could be simultaneously amplified and fluorescently labeled. To establish the system, 12 pre-characterized fungal strains were analyzed; and the method was validated by using 21 clinical isolates as blinded samples. As the microarray was able to detect and clearly identify the fungal pathogens within 4 h after DNA extraction, this system offers an interesting potential for clinical microbiology laboratories.     

Use of an oligonucleotide array for laboratory diagnosis of bacteria responsible for acute upper respiratory infections

We developed a diagnostic array of oligonucleotide probes targeting species-specific variable regions of the genes encoding topoisomerases GyrB and ParE of respiratory bacterial pathogens. Suitable broad-range primer sequences were designed based on alignment of gyrB/parE sequences from nine different bacterial species. These species included Corynebacterium diphtheriae, Fusobacterium necrophorum, Haemophilus influenzae, Legionella pneumophila, Moraxella catarrhalis, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. Specific probe sequences were selected by comparative analysis against the European Bioinformatics Database, as well as gyrB/parE sequences generated for this study. To verify specificity, at least six initial oligonucleotide probe sequences per bacterial species were tested by hybridization on a solid glass support using culture collection strains as templates. Finally, three oligonucleotide probes per bacterial species were utilized to examine 65 middle ear fluid and 29 throat swab samples. The sensitivities of the developed assay compared to classic culture from middle ear fluid samples for H. influenzae, M. catarrhalis, and S. pneumoniae were 96 (93 for culture), 73 (93 for culture), and 100% (78% for culture), respectively. No cross-reactivity with bacterial species belonging to the normal oral flora was observed when the 29 throat swab samples were studied. The sensitivity of the assay to detect S. pyogenes from these samples was 93% (80% for culture). These results provide a proof of concept for the diagnostic use of microarray technology based on broad-range topoisomerase gene amplification, followed by hybridization and specific detection of bacterial species.     

Microarray assay for detection and discrimination of Orthopoxvirus species

A microarray method was developed for simultaneous detection and identification of six species of Orthopoxvirus (OPV) including Variola, Monkeypox, Cowpox, Camelpox, Vaccinia, and Ectromelia viruses. The method allowed us to discriminate OPV species from varicella-zoster virus (VZV), Herpes Simplex 1 virus (HSV-1), and Herpes Simplex 2 virus (HSV-2) that cause infections with clinical manifestations similar to OPV infections. The nucleotide sequences of the C23L/B29R and the B19R genes identified for 86 and 72 different OPV strains, respectively, were used to design species-specific microarray oligonucleotide probes (oligoprobes). The microarray also contained several oligoprobes selected from the ORF31, US4, and US5 genes of VZV, HSV-1, and HSV-2, respectively. The samples (from HSVs or OPVs) of ssDNAs for analyses were prepared by using asymmetric PCR followed by chemical labeling of ssDNA with Cy3 dye. DNA from 52 samples of various OPV species, two isolates of VZV, two of HSV-1, and three of HSV-2 were tested using the developed microarray assay; all tested viruses were accurately identified. To ensure the robustness of the microarray assay, three additional unrelated variola virus strains with unknown sequences of the C23L/B29R and the B19R genes were tested. In each instance the microarray unambiguously identified them as Variola virus species. The results obtained in this study demonstrated that this new microarray method is a valuable tool for the rapid and accurate detection and differentiation of these important viral pathogens.     

High-throughput method for detecting genomic-deletion polymorphisms

DNA microarrays have been successfully used with different microorganisms, including Mycobacterium tuberculosis, to detect genomic deletions relative to a reference strain. However, the cost and complexity of the microarray system are obstacles to its widespread use in large-scale studies. In order to evaluate the extent and role of large sequence polymorphisms (LSPs) or insertion-deletion events in bacterial populations, we developed a technique, termed deligotyping, which hybridizes multiplex-PCR products to membrane-bound, highly specific oligonucleotide probes. The approach has the benefits of being low cost and capable of simultaneously interrogating more than 40 bacterial strains for the presence of 43 genomic regions. The deletions represented on the membrane were selected from previous comparative genomic studies and ongoing microarray experiments. Highly specific probes for these deletions were designed and attached to a membrane for hybridization with strain-derived targets. The targets were generated by multiplex PCR, allowing simultaneous amplifications of 43 different genomic loci in a single reaction. To validate our approach, 100 strains that had been analyzed with a high-density microarray were analyzed. The membrane accurately detected the deletions identified by the microarray approach, with a sensitivity of 99.9% and a specificity of 98.0%. The deligotyping technique allows the rapid and reliable screening of large numbers of M. tuberculosis isolates for LSPs. This technique can be used to provide insights into the epidemiology, genomic evolution, and population structure of M. tuberculosis and can be adapted for the study of other organisms.     

腹泻相关致病菌基因芯片的制备及应用

目的确定引起人类感染性腹泻的11种病原微生物,并制备芯片用于检测门诊腹泻患者粪便标本中的致病菌。方法根据本院2009年1月至2012年12月期间腹泻门诊的粪便病原菌检测数据,采用生物信息学的方法,收集11种病原菌的所有基因序列,设计引物及探针,优化并制备芯片,PCR扩增杂交并对杂交结果进行分析。用该芯片对本院保存的163个肠道致病菌临床分离株进行鉴定来评价芯片的特异性,用芯片来检测掺有不同浓度沙门氏菌的粪便标本评价芯片的灵敏度。同时收集2010年6月至2013年3月在本院就诊的1052份腹泻患者粪便标本,平行进行PCR扩增、细菌培养、基因芯片检测,比较不同检测方法的阳性率。结果成功制备了腹泻相关11种致病菌检测芯片。应用制备的芯片检测了163个临床分离株,准确率达100％。与PCR方法比较,基因芯片检测沙门氏菌的灵敏度达102CFU／ml,比PCR法检测灵敏度高10倍。用该芯片对临床1052份腹泻患者腹泻标本进行检测,与传统的培养法及PCR法比较,有较高的阳性检出率,达36％,比常规细菌培养阳性率高13％（X2=2．28,P〈0．05）,比PCR检出率高4％（X2=5．16,P〉0．05）。结论成功制备11种腹泻相关致病菌基因芯片,能同时对11种腹泻致病菌进行检测,有较好的特异性和灵敏度,有更高的阳性率,可以用于临床检测。     

Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR

A multiplex PCR method was developed to identify simultaneously multiple fungal pathogens in a single reaction. Five sets of species-specific primers were designed from the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the rRNA gene to identify Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus. Another set of previously published ITS primers, CN4 and CN5, were used to identify Cryptococcus neoformans. Three sets of primers were used in one multiplex PCR to identify three different species. Six different species of pathogenic fungi can be identified with two multiplex PCRs. Furthermore, instead of using templates of purified genomic DNA, we performed the PCR directly from yeast colonies or cultures, which simplified the procedure and precluded contamination during the extraction of DNA. A total of 242 fungal isolates were tested, representing 13 species of yeasts, four species of Aspergillus, and three zygomycetes. The multiplex PCR was tested on isolated DNA or fungal colonies, and both provided 100% sensitivity and specificity. However, DNA from only about half the molds could be amplified directly from mycelial fragments, while DNA from every yeast colony was amplified. This multiplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify common clinical fungal isolates.