DNA Multiplex PCR Research Articles

A novel multiplex PCR assay to detect and distinguish between different types of Paenibacillus larvae and Melissococcus plutonius, and a survey of foulbrood pathogen contamination in Japanese honey.

Paenibacillus larvae and Melissococcus plutonius are the causative agents of American and European foulbroods of honey bees, respectively. Since their virulence and resistance to disinfectants differ depending on the genotypes/phenotypes of the strains, the discrimination of strain types is important for the effective control of these diseases. Methods to detect and differentiate pathogens in honey are useful for surveying the contamination status of beehives/apiaries. In the present study, we selected a sequence (GenBank accession no. FI763267) as the specific target for enterobacterial repetitive intergenic consensus (ERIC) II-type P. larvae strains for the first time and developed a novel multiplex PCR assay that precisely distinguishes between the major types of foulbrood pathogens (ERIC I and II P. larvae and typical and atypical M. plutonius) in one reaction. In addition, we found that commercially available kits designed for DNA extraction from Mycobacterium in feces efficiently extracted DNA from foulbrood pathogens in honey. Using the multiplex PCR assay and DNA extraction kits, all the targeted types of P. larvae and M. plutonius were detected in honey spiked with the pathogens at a concentration of 100 bacterial cells/strain/ml. Moreover, 94% of the Japanese honey samples examined in the present study were contaminated with one or more types of the foulbrood pathogens. These results indicate that the newly developed methods are useful for detecting foulbrood pathogens in honey. The epidemiological information obtained by these methods will contribute to the effective control of foulbroods in apiaries.

Identification of animal derivatives contained in commercial chicken feeds using multiplex-PCR

Availability of commercial feeds for chicken force farmers to use them due to the practicality than hand formulated feed. Cannibalism and spread of infectious disease may be occurred by feed without knowing its contents. Therefore, the aim of this study was to determine species contained in commercial feeds for chicken using multiplex-PCR mitochondrial DNA Cytochrome b gene. This study used five commercial feeds for chicken produced by five different companies as samples. They were namely PK1, PK2, PK3, PK4, and PK5. Hand-made meat meals from goat, chicken, bovine, and pig were used as positive controls. Genomic DNA was extracted from those samples, and they were used as DNA template for PCR. The PCR products were then visualized using 2% agarose gels under the UV light. The results showed that some samples were positively containing animal derivatives. The PK1 sample contained chicken, bovine, and pig which are respectively indicated by 227, 274, and 398 bp of DNA bands. Moreover, PK4 and PK5 samples contained bovine species. On the other hand, PK2 and PK3 samples did not contain animal derivatives, especially four species used as markers in this study. It can be concluded that animal derivatives have been detected in three commercial feeds for chicken using the multiplex-PCR technique. This study recommended avoiding a commercial feed for chicken since it contained chicken derivatives to prevent cannibalism.

Detection of rare allele in Indian population leading to solution of disputed paternity case: A case study

There are a variety of multiplex PCR DNA amplification kits available in the market which are being used in forensic applications for the purpose of individualization. All these amplification kits are supposed to be validated for world population, hence could be utilized universally for human identification and establishment of DNA Database worldwide. The allelic ladder provided by the manufacturers supplied along with these kits covers almost all the possible range of alleles in a given locus and are hence considered to be suitable for genetic studies worldwide.

Prevalence of Antibiotic-Resistant Escherichia coli Isolates from Local and Imported Retail Chicken Carcasses

The spread of antibiotic resistance among bacterial strains has been associated with consumption of food contaminated with both pathogenic and nonpathogenic bacteria. The objective of this study was to determine the prevalence of antibiotic resistant Escherichia coli isolates in local and imported retail raw chicken meat in Qatar. A total of 270 locally produced (chilled) and imported (chilled or frozen) whole chicken carcasses were obtained from three Hypermarket stores in Qatar. The 216 E. coli isolates recovered from the chicken samples were subjected to antibiotic susceptibility testing with the disk diffusion method. Extended-spectrum β-lactamase (ESBL) production was evaluated with the double disk synergy test. Isolates harboring colistin resistance were identified with a multiplex PCR assay and DNA sequencing. Nearly 89% (192) of the 216 isolates were resistant to at least one of the 18 antibiotics tested. Isolates from local and imported chicken carcasses had relatively higher resistance to sulfamethoxazole (62% of isolates), tetracycline (59.7%), ampicillin and trimethoprim (52.3% each), ciprofloxacin (47.7%), cephalothin (45.4%), and colistin (31.9%). Less resistance was found to amoxicillin-clavulanic acid (6%), ceftriaxone (5.1%), nitrofurantoin (4.2%), piperacillin-tazobactam (4.2%), cefepime (2.3%), meropenem (1.4%), ertapenem (0.9%), and amikacin (0.9%). Nine isolates (4.2%) were ESBL producers, and 137 (63.4%) were multidrug resistant. The percentages of multidrug-resistant, ESBL-producing, and colistin resistant isolates were significantly higher among isolates from local chilled than from imported chilled and frozen chicken samples. Our findings indicate the high prevalence of antibiotic-resistant E. coli in chicken meat sold at retail in Qatar.

Detection of Epstein-Barr virus DNA in saliva of HIV-1-infected individuals with oral hairy leukoplakia.

We present three cases of oral hairy leukoplakia (OHL) in whom the diagnosis was established by EBV DNA detection in whole saliva. Three HIV-infected patients came to the Oral Medicine Clinic with similar chief complaints of asymptomatic white lesions on the tongue. All patients were diagnosed with suspected OHL and oral thrush also in the first patient. A multiplex PCR DNA microarray was performed to detect EBV DNA in saliva collected by spitting method. All saliva samples showed positive results for EBV DNA, and the definitive diagnosis of OHL was made. Resolution of lesions was found at 1- to 2-month follow-up after treatment with application of acyclovir 5% cream 5 times daily. Additionally, anti-fungal treatment was given to the first patient and anti-retroviral treatment to the first and second patients. EBV is mostly transmitted by asymptomatic shedding into saliva. Therefore, the detection of salivary EBV DNA is useful in establishing a definitive diagnosis of OHL allowing more effective treatment for both HIV-infected patients receiving ART and treatment-naïve patients at any CD4+count.

Simultaneous Detection of Multiple Wine-Spoilage Organisms Using a PCR-Based DNA Dipstick Assay.

Background: The presence of microbial contaminants such as Brettanomyces in wine can lead to undesirable wine. Therefore, monitoring for the presence of these spoilage organisms is critical for winemakers to ensure the quality of their end product. Objective: To address this problem, Molecular Epidemiology, Inc. (MEI, Seattle, WA) has developed a wine-spoilage organism detection kit consisting of a multiplex PCR DNA dipstick that simultaneously detects these organisms. Methods: Wine samples obtained from local wineries that tested negative by routine microbiological culture were spiked with the target microorganisms, while samples that were designated as spoiled by the wineries were used as-is without spiking for assessing the performance characteristics of the DNA dipstick assay. Microbial enumeration was performed following standard microbiological plating methods. Samples spiked with low cell numbers (<5 cells per 100 mL) were enriched using wine enrichment media (WSE; optional component of the kit) prior to analysis using the DNA dipstick assay. Suitability of WSE medium to support the growth of wine-spoilage microorganisms was compared with standard microbiological media. Results: Testing of 92 diverse bacterial and yeast strains commonly found in winery and food operations and 50 various strains of spoilage organisms isolated from wineries indicated that the dipstick assay can exclusively detect the target wine-spoilage microorganisms. All target spoilage organisms in samples containing low cell numbers (<5 cells per 100 mL) were detected by dipstick assay 48 h postenrichment in WSE, except for a few strains of Brettanomyces bruxellensis that required longer incubation times. Conclusions: The wine-spoilage organism detection kit has a detection limit of 10 cells/mL. Highlights: The kit can be used at different stages of the wine-making process to detect multiple spoilage-causing microorganisms in a single assay, thus offering a convenient test system for winemakers interested in monitoring the quality of their product.

Dipstick Assay for Rapid Detection of Beer Spoilage Organisms

Background: Beer spoilage caused by wild yeast and bacteria is a major concern to both commercial and home brewers. Objective: To address this problem, Molecular Epidemiology Inc. (MEI, Seattle, WA) has developed a beer spoilage organism detection kit consisting of an enrichment media (BSE) and a multiplex PCR DNA dipstick that simultaneously detects these organisms within 2h following enrichment. Methods: The kit was tested by using samples obtained from breweries located in the Greater Seattle area. Samples were spiked with the target microbes, when necessary, and used for assessing the performance characteristics of the DNA dipstick assay. Microbial enumerations were performed as per the standard microbiological plating methods. The suitability of the BSE medium to support the growth of beer spoilage microbes was compared with the industry-approved NBB-C medium (Dohler, Darmstadt, Germany). Results: Inclusivity (a panel of 50 isolates) and Exclusivity (a panel of 92 isolates) testing indicated that the dipstick assay can exclusively detect the indicated target beer spoilage microbes. When compared with the NBB-C medium (Dohler, Darmstadt, Germany) approved by the European Brewers Convention for beer spoilage organisms, the BSE medium supported faster growth of critical spoilage lactic acid bacteria such as Lactobacillus brevis, L. lindneri, and Pediococcus damnosus. Conclusions: The beer spoilage organism detection kit has a detection limit of 10 cells/mL. Highlights: The kit can be used at different stages of the brewing process, thus offering a convenient, cost effective, and faster test system for brewers interested in monitoring the quality of their product.

IDENTIFICAÇÃO MOLECULAR DE OSTRAS NATIVAS NA COSTA DO MARANHÃO, BRASIL

As ostras são encontradas ao longo de toda a costa do Brasil. Os fenótipos das espécies variam consideravelmente de acordo com as caracterí­­sticas do habitat. O presente estudo investigou a existência de diferentes espécies de ostra do gênero Crassostrea no litoral do Maranhão, utilizando a técnica de PCR Multiplex e DNA barcoding. Os resultados da PCR Multiplex revelaram duas bandas distintas, caracterí­­sticas das espécies C. gasar e C. rhizophorae num total de 135 amostras analisadas. No sequenciamento do gene COI de 98 amostras obteve-se fragmentos de 695 pb e 15 haplótipos para C. gasar e de 640 pb e oito haplótipos para C. rhizophorae. A árvore haplótipica agrupou fortemente as duas espécies em clados diferentes com 100% de bootstrap. Divergências intraespecí­­ficas foram de 0,2% para ambas, e a interespecí­­fica de 23,6%. A similaridade das sequencias deste estudo com sequências presentes no BoldSystems variou de 97,01 a 98,37 para C. rhizophorae e de 97,55 a 99,84 tanto para C. gasar como para C. brasiliana, reforçando uma problemática na taxonomia do grupo, que apontam para a sinonimia dessas espécies. Portanto, o DNA barcoding permitiu identificar as amostras coletadas e confirmou a existência de duas espécies de ostra

Multiplex PCR effectively identifies tetraploid Triticum AABB – or AAGG-genome species

AbstractWe developed a multiplex PCR DNA marker for quick and easy identification of the AAGG-genome timopheevii lineage, including Triticum timopheevii, Triticum araraticum and hexaploid Triticum zhukovskyi (AAAmAmGG), and the AABB-genome emmer wheat lineage, including Triticum durum, Triticum dicoccum and Triticum dicoccoides. Distinguishing between tetraploid AAGG- and AABB-genome wheat species based on morphology is known to be difficult. This multiplex PCR system is based on the simultaneous PCR amplification of two chloroplast regions, matK and rbcL. The matK region molecularly distinguishes the two lineages, whereas the rbcL region is a positive control amplicon. We also examined whether the simple sequence repeat is a fixed mutation within species, using genetic resources in the collection of KOMUGI, Kyoto University, which comprises accessioned species collected across diverse geographical areas. The multiplex PCR marker distinguished AAGG from AABB species with complete accuracy.

Molecular Detection of Mycobacterium Tuberculosis Complex from Sputum of Clinically Suspected Tuberculosis Cases

Background: Tuberculosis (TB) is a communicable disease and it ranks second of all infectious agents due to co-infection with HIV . The causative agent of tuberculosis is a group of mycobacteria known as Mycobacterium tuberculosis complex. Mycobacterium tuberculosis complex consist of M. tuberculosis, M. bovis, M. africanum, M. microti, M. canetti. In PCR study, Most commonly sensitivity is higher in smear positive samples (95-100%) rather than smear negative specimens (46-63%). OBJECTIVE: To detection of Mycobacterium tuberculosis complex by Line Probe Assay. Methods: The study was done from non-interventional approaching study of 50 suspected patients of tuberculosis had visited the TB chest/ DOTS centre. Sputum sample collected early morning in a wide- mouth container from the patients having history of cough more than 2 weeks. Methodology used Z-N staining and for detection of MTB complex was done using MTBDR plus assay, multiplex PCR DNA strip assay (Hain Lifescience, Nehren, Gernamy) which is commercially available. M. tuberculosis secreted an important protein is MPT64, a 24-kDa protein. The major culture filtrate protein (24-kDa) is MPT64 encoded by the RD2 region genes and has been exposed to be an exact antigen that differentiates the M. tuberculosis complex from the mycobacteria other than tuberculosis (MOTT) Species. Results: In 50 samples, out of which 10 (20%) were smear positive & 40(80%) were smear negative. Out of 10 smear positive, 9 (90%) were MTB (Mycobacterium tuberculosis) & 01 (10%) was NTM (Non-tuberculous Mycobacteria) by PCR method. Out of 40 smear negative, 30 (75%) were positive by PCR method. Out of 30, 28(93%) were MTB & 02(7%) were NTM. Rests of the 10(25%) samples were found negative for M. tuberculosis complex. Conclusions: This study proved that PCR is a specific and sensitive method in comparison of sputum microscopy after staining with Z-N technique and it helpful the clinicians ability to diagnose and treat the patients on time. This will ensure early treat to patients and prevent further transmission of disease.

Role of golden jackals (Canis aureus) as natural reservoirs of Dirofilaria spp. in Romania.

BackgroundDirofilaria immitis and Dirofilaria repens are mosquito-transmitted zoonotic nematodes, causing heartworm disease and skin lesions, respectively, in carnivores. In Europe, the domestic dog is apparently the main definitive host, but patent infections occur also in other species of carnivores. The rapid spread of the golden jackals (Canis aureus) throughout Europe opens a question of involvement of this species in the sylvatic cycle of pathogens in the colonised territories, including Dirofilaria spp.MethodsBetween January 2014 and May 2015, 54 golden jackals from 18 localities in Romania were examined by full necropsy for the presence of adult filarioid nematodes and blood samples from all animals were screened for the presence of microfilariae of D. immitis, D. repens and Acanthocheilonema reconditum by multiplex PCR DNA amplification.ResultsNematodes morphologically identified as D. immitis were found in 18.52 % of the animals, originating from the southern part of Romania. No D. repens or A. reconditum were found at necropsy. The molecular prevalence in blood samples from the same animals was 9.26 % for D. immitis and 1.85 % for D. repens. All samples were negative by PCR for A. reconditum.ConclusionThe relatively high prevalence of Dirofilaria spp. infections in golden jackals from Romania together with the increasing density of the jackal populations highlight their potential role in the transmission of these zoonotic parasites and in the maintenance of natural disease foci.

Association of CD40 gene polymorphisms and serum CD40 levels with the pathogenesis of systemic lupus erythematosus.

Objective To explore the association of CD40 gene single nucleotide polymorphisms (SNPs) and haplotypes with the susceptibility to systemic lupus erythematosus (SLE), as well as the association of serum levels and genotypes of CD40 with the occurrence of SLE. Methods A multiplex PCR single-base extension assay (PCR-SBE) and DNA sequencing were performed to analyze 4 SNPs of the CD40 gene, including rs1883832 C/T, rs13040307 C/T, rs752118 C/T and rs3765459 G/A, in 205 patients with SLE (SLE group) and 220 healthy human controls (control group). Enzyme-linked immunosorbent assay (ELISA) was conducted to measure serum levels of CD40 in these subjects. Results Compared with the control group, the SLE group showed significantly increased serum levels of CD40(P < 0.05). There were significant differences in genotype and allele frequencies of the SNP rs1883832 C/T in the CD40 gene between the SLE group and control group (all P < 0.01). Relative risk analysis showed that the risk of developing SLE in rs1883832 T allele carriers was 1.517 times that in rs1883832 C allele carriers (OR = 1.517, 95% CI: 1.157-1.990, P = 0.003). Moreover, serum levels of CD40 were significantly higher in rs1883832 T allele carriers than in rs1883832 C allele carriers (P < 0.01). The risk of developing SLE was significantly increased in TCCA haplotype carriers compared with the healthy controls (OR = 2.322, 95% CI: 1.181 -4.564, P = 0.012). Conclusion The CD40 gene rs1883832 C/T polymorphism and its TCCA haplotype were both associated with the occurrence of SLE, and the rs1883832 T allele may be a gene predisposing to SLE. Key words: Lupus erythematosus, systemic; Antigens, CD40; Polymorphism, single nucleotide; Haplotypes; Genotype; Alleles

The evaluation of a multiplex PCR-DNA dipstick assay with culture method and EZ Typhi Carrier DNA assay to detect Salmonella Typhi in well water samples

IntroductionSalmonella Typhi is the causative agent for typhoid fever that infects people especially in underdeveloped and developing countries. Even though culture method is the gold standard for detection of this pathogen, there are some limitations such as time consuming and less sensitive that leads to the development of a more sensitive and efficient method such as polymerase chain reaction (PCR). ObjectiveTo evaluate the sensitivity of a multiplex PCR-DNA dipstick assay incomparison with culture method and EZTyphi Carrier DNA assay for the detection of S.Typhi in well water samples. MethodsA total of 580 well water samples were collected and filtered using filter membrane. Bacteria trapped on the membrane was enriched in a selective Selenite F and evaluated using these three methods; (i) Culture method: Bacteria culture was plated on a selective media agar plate and further tested by biochemical tests and serotyping (test was performed within 3 days); (ii) EZTyphi Carrier DNA assay: Extracted DNA from bacteria culture was amplified using two sets of primers targeted for S. Typhi (1238 bp) and internal amplification control (IAC) (810 bp) (3 hours); (iii) Multiplex PCR-DNA dipstick assay: Extracted DNA was amplified using four sets of primers targeted for S. Typhi (70 bp), S. Paratyphi A (93 bp), pan-Salmonella (146 bp) and IAC (123 bp) (2 hours). Results & DiscussionFifteen out of 580 water samples were positive S.Typhi by multiplex PCR-DNA dipstick assay while one sample was positiveS. Typhi by EZTyphi Carrier DNA and culture method. This finding proved that the multiplex PCR-DNA dipstick was superior to culture method and PCR-agarose gel electrophoresis since it has higher positivity compared to the other two methods. ConclusionA rapid and sensitive multiplex PCR-DNA dipstick assay can be used to detect S. Typhi in well water especilally in typhoid endemic area.

Detection systems for carbapenemase gene identification should include the SME serine carbapenemase

Carbapenemase detection has become a major problem in hospitals that encounter outbreaks of infections caused by carbapenem-resistant Gram-negative bacteria. Rapid detection systems have been reported using multiplex PCR analyses and DNA microarray assays. Major carbapenemases that are detected by these systems include the KPC and OXA serine carbapenemases, and the IMP, VIM and NDM families of metallo-β-lactamases. However, increasing numbers of the SME serine carbapenemase are being reported from Serratia marcescens, especially from North and South America. These organisms differ from many of the other carbapenemase-producing pathogens in that they are generally susceptible to the expanded-spectrum cephalosporins ceftazidime and cefepime while retaining resistance to almost all other β-lactam antibiotics. Thus, multiplex PCR assays or DNA microarray testing of carbapenem-resistant S. marcescens isolates should include analyses for production of the SME carbapenemase. Confirmation of the presence of this enzyme may provide reassurance that oxyimino-cephalosporins can be considered for treatment of infections caused by these carbapenem-resistant pathogens.

An in-house multiplex PCR for detection of S. typhi and S. paratyphi A: A comparison between detection method by agarose gel electrophoresis and lateral flow assay

Background: Typhoid and paratyphoid still remain as public health problem in developing and underdeveloped countries caused by Salmonella Typhi and Salmonella Paratyphi. Culture method is the gold standard to diagnose typhoid and paratyphoid fever as well as carriers. However, the method is less sensitive and produces results within 2-7 days. Thus, the study aims to develop the multiplex PCR for simultaneous detection of S.Typhi, S.Paratyphi A and Salmonella genus in presence of internal amplification control (IAC) with detection method via agarose gel electrophoresis and compare with lateral flow assay. Methods: PCR was optimized with annealing temperature ranging between 55-75̊C. Other parameters such as concentration of labeled primers, MgCl2, dNTPs and Taq polymerase were also optimized and the detection was performed via 2% agarose gel electrophoresis. For lateral flow assay, the nitrocellulose membrane was dotted with biotin, anti-FITC, anti-Cy5, anti-Dig and anti-DNP and assembled with conjugate pad. The analytical sensitivity was carried out by agarose gel electrophoresis and lateral flow assay. Each amplicon was applied onto the dotted dipstick and the intensity of the dots was observed in 15 min. The test was validated with stool samples spiked with 25S. Typhi, 25 S. Paratyphi A and 25 otherSalmonella serovars and other bacteria. The test was also validated with nine stool samples of food handlers. Results: The multiplex PCR successfully developed to detect S. Typhi, S. Paratyphi A, Salmonella genus and IAC without any cross-reaction. The detection limit for S. Typhi and S. Paratyphi A were 0.16 ng/ml and 0.08 ng/ml respectively using lateral flow assay compared 0.63 nl/ul for both S. Typhi and S. Paratyphi A using agarose gel electrophoresis. The test was successfully validated with spiked stool samples of 75 bacterial isolates and 9 stool samples of food handlers during typhoid outbreak with both sensitivity and specificity of 100%. Conclusion: The findings suggest that the multiplex PCR-DNA dipstick is a potential assay as molecular diagnostics for detection of S. Typhi and S. Paratyphi A.

Predicting Viruses Accurately by a Multiplex Microfluidic Loop-Mediated Isothermal Amplification Chip

Multiplex gene assay is a valuable molecular tool not only in academic science but also in clinical diagnostics. Multiplex PCR assays, DNA microarrays, and various nanotechnology-based methods are examples of major techniques developed for analyzing multiple genes; none of these, however, are suitable for point-of-care diagnostics, especially in resource-limited settings. In this report, we describe an octopus-like multiplex microfluidic loop-mediated isothermal amplification (mμLAMP) assay for the rapid analysis of multiple genes in the point-of-care format and provide a robust approach for predicting viruses. This assay with the ability of analyzing multiple genes qualitatively and quantitatively is highly specific, operationally simple, and cost/time-effective with the detection limit of less than 10 copies/μL in 2 μL quantities of sample within 0.5 h. We successfully developed a mμLAMP chip for differentiating three human influenza A substrains and identifying eight important swine viruses.

Comparison of Multiplex PCR and Acid Fast and Auramine -Rhodamine Staining for Detection of Mycobacterium tuberculosis and Non tuberculosis Mycobacteria in Paraffin- Embedded Pleural and Bronchial Tissues with Granulomatous Inflammation and Caseous Necrosis

Objective The aim of this study was to compare the sensitivity and specificity of Acid fast and Auramine-Rhodamine staining and Multiplex PCR for the detection of Mycobacterium tuberculosis complex and non tuberculosis Mycobacteria on formalin fixed paraffin embedded tissues (FFPE) Materials and Methods Forty cases of FFPE pleural and bronchial tissue with chronic granulomatous inflammation and caseous necrosis and 10 cases with bronchogenic carcinoma as controls were investigated. We designed a Multiplex PCR DNA amplification method with two targets: 123bp DNA fragment from IS6110, which is present only in mycobacterium tuberculosis complex and 162bp DNA encoding Ag 85complex which is present in all of mycobacteria. The FFPE also stained by Acid fast and Rhodamine-Auramine staining method. Results In 26 samples (65%) 123 bp and 162 bp DNA fragments were detected together (12 in bronchial samples and 14 in pleural samples).The 162 bp fragment wasn't detected alone. The sensitivity of PCR was 65% and the specificity was 100%. Eleven cases were positive for Acid fast staining. There was 27.5% sensitivity and 100% specificity. Thirteen cases were positive for Auramine-Rhodamine staining (A-R-S); there was 32.5% sensitivity and 100% specificity. All of the 10 controls were negative for 123 bp, 162 bp DNA fragments, for Acid fast and Auramine-Rhodamine staining. Conclusion Multiplex PCR is a sensitive, specific and rapid method for detection of M. tuberculosis in FFPE tissues.

Detection of Genetically Modified Crops by Combination of Multiplex PCR and Low-density DNA Microarray

To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified (GM) soybean. Seventeen capture probes (PCR products) and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean (GTS40-3-2), maize (Mon810, Nk603, GA21), canola (T45, MS1/RF1), and rice (SCK) in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean (Roundup Ready GTS40-3-2) and canola (MS1/RF1). A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity. A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

DNA Microarray-Based Detection and Identification of Fungal Pathogens in Clinical Samples from Neutropenic Patients

The increasing incidence of invasive fungal infections (IFI) in immunocompromised patients emphasizes the need to improve diagnostic tools. We established a DNA microarray to detect and identify DNA from 14 fungal pathogens (Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Candida albicans, Candida dubliniensis, Candida glabrata, Candida lusitaniae, Candida tropicalis, Fusarium oxysporum, Fusarium solani, Mucor racemosus, Rhizopus microsporus, Scedosporium prolificans, and Trichosporon asahii) in blood, bronchoalveolar lavage, and tissue samples from high-risk patients. The assay combines multiplex PCR and consecutive DNA microarray hybridization. PCR primers and capture probes were derived from unique sequences of the 18S, 5.8S, and internal transcribed spacer 1 regions of the fungal rRNA genes. Hybridization with genomic DNA of fungal species resulted in species-specific hybridization patterns. By testing clinical samples from 46 neutropenic patients with proven, probable, or possible IFI or without IFI, we detected A. flavus, A. fumigatus, C. albicans, C. dubliniensis, C. glabrata, F. oxysporum, F. solani, R. microsporus, S. prolificans, and T. asahii. For 22 of 22 patients (5 without IFI and 17 with possible IFI), negative diagnostic results corresponded with negative microarray data. For 11 patients with proven (n = 4), probable (n = 2), and possible IFI (n = 5), data for results positive by microarray were validated by other diagnostic findings. For 11 of 11 patients with possible IFI, the microarray results provided additional information. For two patients with proven and probable invasive aspergillosis, respectively, microarray results were negative. The assay detected genomic DNA from 14 fungal pathogens from the clinical samples, pointing to a high significance for improving the diagnosis of IFI.

Rapid Genotypic Detection of Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis Directly in Clinical Specimens

A multiplex PCR DNA strip assay (Genotype MTBDR) designed to detect rifampin (rpoB) and high-level isoniazid (katG) resistance mutations in Mycobacterium tuberculosis isolates was optimized for clinical specimens. Successful genotypic results were achieved with 36 of 38 (95%) smear-positive respiratory specimens, allowing rapid therapeutic adjustments in transmittable drug-resistant tuberculosis.