Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with "universal" primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.     

Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic Mollicutes Strains by Reverse Line Blot Hybridization

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.     

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5'' amine modified to allow fixation to the membrane. Primers are 5'' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours).The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.Published applications of mPCR/RLB include detection of antibiotic resistance genes^1,2, typing of methicillin-resistant Staphylococcus aureus^3-5 and Salmonella sp⁶, molecular serotyping of Streptococcus pneumoniae^7,8, Streptococcus agalactiae⁹ and enteroviruses^10,11, identification of Mycobacterium sp¹², detection of genital^13-15 and respiratory tract¹⁶ and other¹⁷ pathogens and detection and identification of mollicutes¹⁸. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.     

Molecular serotype identification of Streptococcus agalactiae of bovine origin by multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay

We used a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay and sequencing of a variable region of the cps cluster to identify serotypes of 140 Streptococcus agalactiae (group B Streptococcus; GBS) isolates from cattle. Only 71 (51%) isolates were typeable using antisera, but molecular serotypes (MS) were assigned to 133 (95%) and 139 (99%) isolates by partial cpsE-cpsF-cpsG sequencing and mPCR/RLB, respectively. Ninety-four isolates (67%) belonged to MS III and most belonged to a molecular serosubtype (msst) III-3, which is uncommon among GBS isolates from humans. Our results demonstrate that cps clusters of bovine GBS differ significantly from those of GBS isolates from humans.     

Multiplex PCR-based reverse line blot assay for simultaneous detection of 22 virulence genes in uropathogenic Escherichia coli

Urinary tract infections (UTIs) are among the most common bacterial infections and are responsible for significant morbidity and health care costs worldwide. The main bacterial cause of uncomplicated UTI is Escherichia coli, which possesses numerous virulence factors (VFs). Many studies of the pathogenesis of E. coli UTI have centered on VF genes. Hence, the development of better molecular assays to study VF genes would facilitate these studies. We developed a highly sensitive and specific multiplex PCR-based reverse line blot (mPCR/RLB) assay to simultaneously detect 22 VF genes of uropathogenic E. coli and then used it to characterize 180 isolates from nonpregnant women of child-bearing age with cystitis and 153 fecal isolates from similar-age healthy women, in regional New South Wales, Australia. The assay accurately identified all VF genes (of the 22 under study) known to be present in 30 previously characterized control strains. The detection limits were 28 ng of DNA from E. coli isolates and 50 CFU/ml in mock-infected urine specimens containing known concentrations of E. coli. Cystitis isolates (compared to the fecal isolates) showed a significantly higher prevalence of 18 individual VF genes and contained significantly more VF genes per isolate (median number, 18.5 versus 6.5 [P = 0.001]). Discordance between paired probes for a given VF gene occurred in several clinical test isolates but no reference strains and among the test isolates was associated with fecal source (10% of VF genes versus 2% for cystitis isolates [P < 0.001]). This novel mPCR/RLB method is a potentially powerful tool for investigating the prevalence and distribution of VFs in E. coli.     

Rapid simultaneous detection and identification of six species Candida using polymerase chain reaction and reverse line hybridization assay

The aim of this study was to develop and evaluate PCR based reverse line blot (RLB) hybridization assay for rapid detection of the most common Candida isolates from clinical specimens. A pair of universal primers targeting the ITS2 region of the gene from 28S rRNA to 5.8S rRNA was designed for PCR amplification of DNA from 6 Candida species (C. albicans, C. tropicalis, C. krusei, C. glabrata, C. parapsilosis, C. dubliniensis), the reverse primer was biotin labeled. PCR products, which were 302-441 bp length, were hybridized with 6 specific oligonucleotides probes immobilized on a nylon membrane. These 6 probes proved specific (they hybridized with only their target molecules). The assay was shown to be sensitive in detecting yeast to a concentration of 10 CFU/ml. This method was used to test 100 isolates and 200 vaginal swabs. The results agreed with those of culture for all but 3 of 100 isolates. Sequencing was performed on these 3 samples and confirmed that the culture results were inaccurate. Our results show the PCR-RLB positive rate (49%) is higher than culture (39%) and smear microscopic screening (27%) (P<0.05). In conclusion, the PCR/RLB developed in this study is specific and offers increased sensitivity compared to culture for the detection of Candida species in swab specimens. Moreover, the improved detection of cases of polycandidal candidiasis is advantageous.     

Detection of Salmonella enterica serovar Typhi (S. Typhi) by selective amplification of invA, viaB, fliC-d and prt genes by polymerase chain reaction in mutiplex format

AIMS: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. METHODS AND RESULTS: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC-d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR. Internal amplification control, which co-amplified with prt primers, was also included in the assay. The results showed that all cultures of Salmonella were accurately identified by the assay with no nonspecific amplification in other cultures. The assay had 100% detection probability when a cell suspension of 10(4) CFU ml(-1) (500 CFU per reaction) was used. Salmonella Typhi bacteria were artificially inoculated in the water and food (milk and meat rinse) samples and detected by mPCR after overnight pre-enrichment in buffered peptone water. No Salmonella bacteria could be detected from water samples collected from the field by mPCR or standard culture method. CONCLUSIONS: The developed mPCR assay provides specific detection of S. Typhi. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid methods for detection of S. Typhi from complex environmental matrices are almost nonexistent. The mPCR assay reported in this study can be useful to identify S. Typhi bacteria in field environmental samples.     

Molecular detection methods developed for a systemic rickettsia-like bacterium (RLB) in Penaeus monodon (Decapoda: Crustacea)

Molecular detection methods were developed to aid in the diagnosis of a rickettsia-like bacterium (RLB) which caused severe mortalities of farm-raised Penaeus monodon in Madagascar. Using primers derived from the 16S rRNA gene of bacteria, a PCR assay was optimized to amplify this region of the genome of the RLB, using extracted DNA from infected P. monodon tissue as the template. The resulting amplified PCR product was sequenced and 2 novel primers were selected from the variable region of the gene. These primers amplified a 532 bp fragment of DNA originating from the rickettsia-infected samples. The PCR assay was optimized and tested on DNA extracted from specific pathogen-free (SPF) P. vannamei tissue and several other strains of bacteria. The PCR assay with the rickettsia-specific primers was specific for this RLB and did not amplify the other DNA samples tested. The 532 bp PCR-amplified fragment was labeled with digoxigenin (DIG) for in situ hybridization assays. This probe was tested on SPF, RLB and bacteria-infected shrimp specimens preserved in Davidson's fixative. The probe was specific for both natural and experimental rickettsial infections. Hybridization with this probe required a stringent temperature of 65 degrees C, otherwise cross-reactivity was observed with other types of bacteria.     

Rapid detection of Mycobacterium tuberculosis in clinical samples by multiplex polymerase chain reaction (mPCR)

Although the multi-copy and specific element IS6110 provides a good target for the detection of Mycobacterium tuberculosis complex by PCR techniques, the emergence of IS6110-negative strains suggested that false negative may occur if IS6110 alone is used as the target for detection. In this report, a multiplex polymerase chain reaction (mPCR) system was developed using primers derived from the insertion sequence IS6110 and an IS-like elements designated as B9 (GenBank accession no. U78639.1) to overcome the problem of detecting negative or low copy IS6110 containing strains of M. tuberculosis. The mPCR was evaluated using 346 clinical samples which included 283 sputum, 19 bronchial wash, 18 pleural fluid, 9 urine, 7 CSF, 6 pus, and 4 gastric lavage samples. Our results showed that the sensitivity (93.1 %) and specificity (89.6 %) of the mPCR system exceeds that of the conventional method of microscopy and culture. The mPCR assay provides an efficient strategy to detect and identify M. tuberculosis from clinical samples and enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.     

Rapid and sensitive detection of major uropathogens in a single-pot multiplex PCR assay

Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. Escherichia coli is the most common cause of UTI accounting for up to 70?% and a variable contribution from Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella pneumoniae. To establish a complete diagnostic system, we have developed a single-tube multiplex PCR assay (mPCR) for the detection of the above-mentioned four major uropathogens. The sensitivity of the assay was found to be as low as 10(2)?cfu/ml of cells. The mPCR evaluated on 280 clinical isolates detected 100?% of E. coli, P. aeruginosa, P. mirabilis and 95?% of K. pneumonia. The assay was performed on 50 urine samples and found to be specific and sensitive for clinical diagnosis. In addition, the mPCR was also validated on spiked urine samples using 40 clinical isolates to demonstrate its application under different strain used in this assay. In total, mPCR reported here is a rapid and simple screening tool that can compete with conventional biochemical-based screening assays that may require 2-3?days for detection.     

Development and evaluation of real-time PCR assay for the detection of Babesia orientalis in water buffalo (Bubalus bubalis, Linnaeus, 1758)

Babesia orientalis is the causative agent of babesiosis in water buffalo (Bubalus babalis, Linnaeus, 1758). In this study, a TaqMan real-time PCR assay was developed for quantitative detection of B. orientalis in water buffalo. Hybridization probe and oligonucleotide primers were designed based on the v4 region of 18S rRNA gene. Detection limit was determined at 2 parasites. Blood samples were collected from experimentally infected water buffalo, as well as from 180 field samples, which were collected from 4 different geographical locations to the north and south of the Yangtse River. The parasite was detected by real-time PCR on day 2 until day 39 post-infection, while reverse line blot (RLB) was on day 6 until day 36 in experimentally infected water buffalo. For the results of 180 field samples, statistical analysis showed no significant difference in relative effectiveness of real-time PCR and RLB. The analysis also indicated that there was no difference in the prevalence of B. orientalis between the regions of south and north of the Yangtse River by both the real-time PCR assay and RLB detection. These results indicated that the parasite infection has spread to the north of the Yangtse River.     

Optimization and evaluation of a multiplex PCR for simultaneous detection of prominent foodborne pathogens of Enterobacteriaceae

Members of the family Enterobacteriaceae are major pathogens associated with gastrointestinal disorders caused by the consumption of contaminated foods. We have developed a multiplex PCR (mPCR) targeting specific genes for simultaneous detection and differentiation of five major Enterobacteriaceae members, namely, Salmonella sp. (invA), Escherichia coli (uidA), Shigella sp. (ipaH), Klebsiella pneumoniae (khe) and Citrobacter freundii (tpl), from both pure cultures and contaminated food samples, along with an internal amplification control (IAC). Simultaneous amplification of these five genes was optimized using reference strains and further evaluated on large number of isolates recovered from clinical and environmental sources. The mPCR assay showed high sensitivity for detecting 10 CFU/PCR for the above-mentioned pathogens directly from serially diluted overnight cultures. The mPCR assay was also able to detect all five pathogens spiked at an initial count of 10 CFU/g of meat and rice samples following an enrichment of 10 h in Brain Heart Infusion broth. To assess the practical application of this mPCR assay, we evaluated its efficacy for detecting possible contamination on natural samples, such as meat, fish, pastries and water. Based on the results, we suggest that this mPCR assay would be of immense help in detecting low counts of important Enterobacteriaceae pathogens inexpensively and thus can be used for the regular monitoring of food quality.     

转基因白桦外源基因的多重PCR快速检测

根据转化的载体序列上T-DNA中的目的基因bt,选择性筛选标记基因nptⅡ和报告基因gus设计三对特异性引物,PCR产物片断大小分别为247、449、668 bp,应用多重PCR (mutiplex-PCR)的方法同步检测18株转基因白桦中三个基因的整合状况;用阳性对照为模板,对单重PCR（simplex-PCR）和多重PCR的各项指标进行比较。结果表明多重PCR检测多个外源基因在敏感性方面与单重PCR相比并没有减弱,而且略有提高;对18株样品的多重PCR同步检测无假阳性出现,结果准确,同时在操作中具有减少污染,缩短时间和节约成本等优点。因此,在对转基因白桦的外源基因的定期检测中,多重PCR是一种非常有效而便捷的方法,可以为转基因的拷贝数,T-DNA旁侧序列特征等转基因整合特性方面的研究提供数据。     

缺失型Duchenne/Becker 肌营养不良家系女性携带者及新发突变的确认

应用多重PCR（multiple polymerase reaction／mPCR）技术,联合DMD基因内部及附近11个短串连重复序列（short tandem repeats,STRs）位点连锁分析,对缺失型Duchenne／Beeker肌营养不良（Duchenne／Becker Muscular Dystrophy,DMD／BMD）家系成员进行DMD基因分型,确定家系中女性成员是否携带者,并进行产前诊断。3个家系中的4名缺失型患者,其中2例为新发突变：4位女性成员中,1名为携带者。应用mPCR和11个STRs的连锁分析,能快速、准确、客观判断家系中女性成员是否携带者身份,适于DMD／BMD临床研究机构遗传咨询、基因诊断和产前诊断常规应用。但在mPCR分析过程中,发现45号外显子扩增产物在不同凝胶中电泳迁移率不同。聚丙烯酰胺凝胶电泳（Polyacrylamide Gels Electrophoresis／PAGE）对mPCR产物分析快速、清晰,但需要注意片段迁移率,以防止分析错误。     

Simultaneous detection of piroplasma infections in field Haemaphysalis qinghaiensis ticks by reverse line blotting

Piroplasmosis is caused by tick-borne haemoprotozoan parasites in the genera of Theileria and Babesia, in which numbers of agents are highly pathogenic for cattle, sheep and goats. We developed a reverse line blot (RLB) assay for detection and differentiation of four different parasites, in which 18S ribosomal RNA gene sequence was amplified with a set of universal primers specific for all members in the genera of Theileria and Babesia; and the probes were designed on the basis of hypervariable region 4 (V4 region) of 18S rRNA gene. Three Theileria and one Babesia can be detected simultaneously on this system and it was sensitive to detect a parasitemia level between 10⁻⁵ and 10⁻⁸%. A total of 149 Haemaphysalis qinghaiensis ticks collected from Lintan County of Gannan Tibetan Autonomous Region was tested by RLB. Among these, 136 tick samples were also tested by a nested PCR assay developed previously. After comparison of these results, it showed that more T. luwenshuni was detected in RLB assay, while more T. uilenbergi was detected in H. qinghaiensis ticks by nested PCR. The RLB has shown capability for simultaneous detection of four species of piroplasm in H. qinghaiensis ticks, indicating its usefulness for epidemiological studies of piroplasmosis.     

Multiplex PCR assay for detection of four major bacterial pathogens causing rainbow trout disease

A multiplex PCR (mPCR) method was designed for the simultaneous detection of 4 major fish pathogens, Flavobacterium psychrophilum, Lactococcus garvieae, Pseudomonas aeruginosa, and P. putida. Each of the 4 pairs of oligonucleotide primers exclusively amplified the 16S rDNA gene of their targeted microorganism. The average detection limits for each organism amplified by mPCR were 2 colony-forming units (CFU) of F. psychrophilum, 3 CFU of L. garvieae, 3 CFU of P. aeruginosa, and 5 CFU of P. putida in mixed cultures. Multiplex PCR did not produce any nonspecific amplification products when tested against 28 related species of bacteria. High amounts of DNA from 1 bacterial species had a significant effect on the amplification sensitivity of the other bacterial species when these were present in lower concentrations in the multiplex reaction. The mPCR assay proved useful for the detection of the bacteria in naturally infected fish. The assay is a sensitive, specific, and reproducible diagnostic tool for the simultaneous detection of 4 pathogenic bacteria that cause disease in fish and offers a potentially useful alternative to the conventional culture-based method.     

Development of multiplex PCR assays based on the 16S-23S rRNA internal transcribed spacer for the detection of clinically relevant nontuberculous mycobacteria

Aims: To accelerate the identification and differentiation of clinically relevant nontuberculous mycobacteria (NTM) with two sets of multiplex PCR (mPCR) targeting the 16S–23S rRNA internal transcribed spacer (ITS) region for timely patient management. Methods and Results: Two mPCR assays were developed: Slow‐Growers (SG) mPCR was used for the detection of slow‐growing mycobacteria, which included Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium gordonae and Mycobacterium xenopi whilst the other mPCR assay labelled as Fast‐Growers (FG) mPCR was used for the detection of Mycobacterium fortuitum complex, Mycobacterium abscessus and Mycobacterium chelonae. In these assays, a common forward primer based on a conserved section of the 16S rRNA region was used in conjunction with species‐specific reverse primers. The mPCRs were tested against 247 clinical mycobacterial isolates and demonstrated 100% specificity and sensitivity. Identification of the mycobacterial species was also validated by DNA sequencing of the 16S–23S ITS region and when further confirmation was needed, hsp65 sequencing was performed. Conclusions: The mPCR assays could be a potentially useful diagnostic tool for the rapid and accurate identification of clinically relevant NTM. Significance and Impact of the Study: In this study, we looked at the frequency of hospital isolated NTM over the last 5 years (2005–2010), and an mPCR targeting the ITS region was developed for NTM species that appeared to be more prevalent in the context of Singapore.     

Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds.     

Rapid and specific identification of Shiga toxin-producing Escherichia coli in faeces by multiplex PCR

AIMS: The object of this study was to develop a multiplex PCR system for rapid and specific identification of Shiga toxin-producing Escherichia coli (STEC) in faeces. METHODS AND RESULTS: A multiplex PCR (mPCR) protocol was developed using a primer pair specific for genes that are involved in the biosynthesis of the O157 E. coli antigen, and primers that identify the sequences of Shiga toxin 1 and 2 (stx 1 and stx1) and the intimin protein (eaeA). The mPCR assay was used for amplification of STEC genes in bacteria directly (after enrichment) in faeces. The test was very sensitive and could detect between 9 and 1 bacterial cells per gram of faeces. The mPCR was used for the examination of 69 bovine faecal samples derived from healthy cattle. The results indicated that 62 x 3% of the samples were positive, generating at least one PCR amplicon of the expected size. CONCLUSIONS: The method can be applied for rapid and specific identification of STEC bacteria in faecal samples, and for differentiation of their main virulence marker genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect Shiga toxin-producing E. coli directly in faeces within a short time represents a considerable advancement over more time-consuming and less sensitive methods for identification and characterization of STEC bacteria.     

四种广普性植物病毒高效mPCR检测方法的建立

本研究建立了能同时检测出烟草花叶病毒(TMV)、黄瓜花叶病毒(CMV)、马铃薯X病毒(PVX)和马铃薯Y病毒(PVY)的多重RT-PCR体系。TMV、CMV、PVX、PVY是四种广普性植物病毒,寄主范围广泛,并且常常发生复合侵染。本研究以上述四种病毒的CP基因部分序列设计引物,以反转录的cDNA为模板,建立多重RT-PCR反应体系,分别扩增出211～417bp的不同长度的基因片断,并通过序列测定来确认扩增序列的特异性。将反转录合成的cDNA进行浓度稀释,来对多重RT-PCR与单重RT-PCR的灵敏度进行比较,结果证明,多重RT-PCR体系能够同时快速检测这四种病毒,并且有很高的灵敏度。