Top PDF PCR-based assay for Mycoplasma hyopneumoniae

PCR based assay for Mycoplasma hyopneumoniae

PCR based assay for Mycoplasma hyopneumoniae

it also con tains a probe that hybridizes to the target nucleic acid sequence ampli?ed by the use of the primer pair of the invention in the polymerase chain reaction.. the [r]

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Detection of Mycoplasma hyopneumoniae in Bronchoalveolar Lavage Fluids of Pigs by PCR

Detection of Mycoplasma hyopneumoniae in Bronchoalveolar Lavage Fluids of Pigs by PCR

The rates of detection of M. hyopneumoniae in pigs with chronic pneumonia obtained by cultivation, immunofluores- cence tests, and PCR (i) with BALFs incubated in Friis me- dium, (ii) with DNA extracted from BALFs, and (iii) with washings from lungs are summarized in Table 2. M. hyopneu- moniae could not be detected by any of the five methods tested in 18 of the 40 pigs investigated. All remaining 22 pigs showed a positive reaction in the PCR with DNA extracted from BALFs. The lungs of 11 of these 22 pigs were also examined by the immunofluorescence test. M. hyopneumoniae could be de- tected in all of them, indicating a complete correspondence of the results of these two methods. Of the 22 pigs showing M. hyopneumoniae by PCR with the DNA extracted from BALF, only 6 had a positive result by the PCR with BALFs incubated in Friis medium. The PCR with postmortem lung washings from 10 of these 22 pigs was positive for 6 pigs. The lowest detection rate was obtained by cultivation of the BALFs, which was positive for only 1 of the 40 pigs investi- gated. M. hyorhinis was isolated from 15 of the 40 pigs. The cultures of the BALFs from 13 pigs could not be evaluated because of the high level of contamination by walled bacteria.
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Diagnosis and Differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis Infections in Pigs by PCR Amplification of the p36 and p46 Genes

Diagnosis and Differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis Infections in Pigs by PCR Amplification of the p36 and p46 Genes

flocculare and M. hyorhinis reduce the specificity of conventional serological detection methods. However, certain antigenic determinants of the p36 and p46 proteins have been shown to be specific for M. hyopneu- moniae. In the present study, pairs of oligonucleotide primers were designed to permit PCR amplification of entire p36 and p46 genes and of internal fragments of these genes. Specific amplicons could be obtained with as low as 0.5 to 50 pg of extracted chromosomal DNA. No amplification product was obtained when testing p36 and p46 primer pairs with genomic DNA or RNA from other mycoplasma species, bacteria, and viruses commonly associated with respiratory diseases in pigs. By using the single p36-PCR method, a positive reaction was demonstrated in 100% (30 of 30) of lungs from pigs that developed typical lesions associated with an M.
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Real Time PCR Assays To Address Genetic Diversity among Strains of Mycoplasma hyopneumoniae

Real Time PCR Assays To Address Genetic Diversity among Strains of Mycoplasma hyopneumoniae

Received 10 December 2007/Returned for modification 16 February 2008/Accepted 26 May 2008 Mycoplasma hyopneumoniae is an important cause of pneumonia in pigs around the world, but confirm- ing its presence in (or absence from) pigs can be difficult. Culture for diagnosis is impractical, and seroconversion is often delayed after natural infection, limiting the use of serology. Numerous PCR assays for the detection of M. hyopneumoniae have been developed, targeting several different genes. Recently, genetic diversity among strains of M. hyopneumoniae was demonstrated. The effect of this diversity on the accuracy and sensitivity of the M. hyopneumoniae PCR assays could result in false-negative results in current PCR tests. In this study, a panel of isolates of M. hyopneumoniae, M. flocculare, M. hyorhinis, and M. hyosynoviae were tested with a number of M. hyopneumoniae-specific PCR assays. Some M. hyopneu- moniae PCR assays tested did not detect all isolates of M. hyopneumoniae. To increase the efficiency of PCR testing, two new real-time PCR assays that are specific and capable of detecting all of the M. hyopneu- moniae isolates used in this study were developed.
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Selective medium for culture of Mycoplasma hyopneumoniae

Selective medium for culture of Mycoplasma hyopneumoniae

2. Materials & methods 2.1. Mycoplasma strains M. hyopneumoniae strains 277/94 and 325/95 were the gift of Niels Friis, Danish Veterinary Institute, Copenhagen. Other strains of M. hyopneumoniae were Danish fi eld isolates from lesions of EP, the gift of Dr Branko Kokotovic, Danish Veterinary Institute, or UK fi eld isolates from slaughterhouse lesions of EP. M. hyorhinis strains were UK fi eld isolates obtained from pig lungs with or without gross lesions at post-mortem. The identity of all organisms was con fi rmed using species-speci fi c PCR amplifying a region of the conserved hypothetical protein mhp165 from M. hyopneumoniae (Baumeister et al., 1998) and the highly conserved 16S rRNA region of M. hyorhinis (Lin et al., 2006). The identity of all M. hyopneumoniae strains was subsequently con fi rmed by whole genome sequencing and genome-wide analysis (J. Welch personal communication).
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Development of a Quantitative Real Time PCR Assay for Detection of Mycoplasma genitalium

Development of a Quantitative Real Time PCR Assay for Detection of Mycoplasma genitalium

Department of Dermatovenereology, Huddinge University Hospital, Karolinska Institutet, S-14 186 Huddinge, Sweden 3 Received 31 January 2005/Returned for modification 3 March 2005/Accepted 9 March 2005 Mycoplasma genitalium is known to cause nonchlamydial, nongonococcal urethritis in men and to be asso- ciated with pelvic inflammatory disease in women. Specific and sensitive PCR methods are needed for diagnosis of this bacterium because it is very difficult to culture from patient samples. To determine the bacterial load in patients’ specimens, a quantitative real-time LightCycler PCR was developed. The housekeeping gene gap encoding glyceraldehyde-3-phosphate dehydrogenase was chosen as the target gene. The assay could consis- tently detect five genome copies per reaction. To evaluate the PCR, we tested 246 selected urethral swab samples from men attending a clinic for sexually transmitted diseases. Eighty-two of the samples were found positive for M. genitalium by a conventional 16S rRNA gene PCR assay, whereas 164 samples were randomly chosen among those tested negative. Of the positive samples, 78 (95.1%) were found positive, whereas 6 (3.7%) of the negatives were found positive by the LightCycler assay. The patient samples were also tested with a quantitative TaqMan assay, and the bacterial load was compared to the LightCycler results. A good linear correlation between the LightCycler and the TaqMan assays was found with a correlation coefficient of 0.89 and a slope of 0.99. Significantly more M. genitalium-positive men had urethritis, discharge, and dysuria than had M. genitalium-negative men. The M. genitalium DNA load in samples from patients with urethritis was significantly higher than in samples from those without (61 and 2.9 copies/ ␮ l, respectively [P ⴝ 0.0005]). This assay may prove useful in the monitoring of treatment and for optimizing sample preparation methods.
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Survey on Mycoplasma hyopneumoniae gilt acclimation practices in Europe

Survey on Mycoplasma hyopneumoniae gilt acclimation practices in Europe

hyopneumoniae positive replacements. In addition, 249 out of 321 (77.6%) farms applied an acclimation process using different strategies, being M. hyopneumoniae vaccination (145 out of 249, 58.2%) and the combination of vaccine and exposure to sows selected for slaughter (53 out of 249, 21.3%) the most commonly used. Notwithstanding, only 53 out of 224 (23.6%) farms, knowing the M. hyopneumoniae initial status and performing acclimation strategies against it, verified the effect of the acclimation by ELISA (22 out of 53, 41.5%), PCR (4 out of 53, 7.5%) or both (27 out of 53, 50.9%). This study showed that three fourths of the farms represented in this European survey have M. hyopneumoniae acclimation strategies for gilts, and one fifth of them verify to some extent the effect of the process. Taking into account that the assessment of acclimation efficacy could help in optimizing replacement gilt introduction into the breeding herd, it seems these practices for M. hyopneumoniae are still poorly developed in Europe.
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Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae

Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae

Abstract Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss to pig producers. Control is achieved by a combination of antimicrobials, vaccination and management practices, but current vaccines offer only partial control and there is a need for improved preventative strategies. A major barrier to advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lack of tools to genetically manipulate the organism. We describe the development and optimisation of the first successful plasmid-based system for the genetic manipulation of M. hyopneumoniae. Our artificial plasmids contain the origin of replication (oriC) of M. hyopneumoniae along with tetM, conferring resistance to tetracycline. With these plasmids, we have successfully transformed M. hyopneumoniae strain 232 by electroporation, generating tetracycline resistant organisms. The persistence of extrachromosomal plasmid and maintenance of plasmid DNA over serial passages shows that these artificial plasmids are capable of self-replication in M. hyopneumoniae. In addition to demonstrating the amenability of M. hyopneumoniae to genetic manipulation and in optimising the conditions necessary for successful transformation, we have used this system to determine the minimum functional oriC of M. hyopneumoniae. In doing so, we have developed a plasmid with a small oriC that is stably maintained over multiple passages that may be useful in generating targeted gene disruptions. In conclusion, we have
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Genetic diversity of Mycoplasma hyopneumoniae isolates of abattoir pigs

Genetic diversity of Mycoplasma hyopneumoniae isolates of abattoir pigs

2011 ), molecular typing of the p146 gene (sequencing and length of VNTR) ( Mayor et al., 2007 ) and multilocus sequence typing (MLST) ( Mayor et al., 2008 ). These studies have observed that patterns obtained from cultured broths and their associated clinical samples were identical, indicating that these techniques could be used without prior cultivation. This study also demonstrates that both MLVA and PCR-RFLP should be considered as reliable tests for quick and rather inexpensive differentiation of M. hyopneumoniae strains without prior isolation. In addition, MLVA typing gave strong evidence that some pigs were infected with multiple strains of M. hyopneumoniae and this was not achieved with the other typing method ( Marois-Cre´han et al., 2012; Vranckx et al., 2011 ). It has been suggested that simultaneous or subsequent infec- tions with more than one strain might result in more severe lung lesions ( Villarreal et al., 2009; Vranckx et al., 2011 ). However, in this study, lungs harboring more than one strain of M. hyopneumoniae as per MLVA results were not associated with higher lesion scores (p = 0.87), indicating that numbers of strains per lungs were not linked with more severe lung lesions in abattoir pigs (data not shown). The MLVA procedure also revealed that the absence of one locus was significantly associated to lower concentrations of bacteria (p < 0.0001) and lower percen- tages of lesions (p < 0.0001), suggesting that this locus of M. hyopneumoniae could be associated with virulence. The gene amplified by this locus encodes for a hypothetical protein of 77–78 kDa (GenBank accessions no. YP 278837.1 of strains J (ATCC 25934), YP 115552.1 of USA 232 and YP 287436.1 of 7448). In a study by Calus et al. (2007) , when comparing the SDS-PAGE patterns from isolates of different herds, the most explicit variability was detected over 74 kDa with one band of 181 kDa specific for two highly virulent isolates. However, attempts to relate total protein profiles according to virulence has been unfruitful ( Calus et al., 2007 ). MLVA and PCR-RFLP clustering of M. hyopneumoniae also revealed that analyzed strains were distributed among all clusters regardless of lesions’ severities, indicating that DNA patterns did not cluster according to virulence. Also, no links were observed between numbers of M. hyopneumoniae cells and severity of lesions.
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MLVA typing of Mycoplasma hyopneumoniae bacterins and field strains

MLVA typing of Mycoplasma hyopneumoniae bacterins and field strains

Although de Castro and others (2006) suggested that developed PCR assays could have suf fi cient sensitivity for M hyopneumoniae typing from clinical samples, a com- plete characterisation by MLVA is not always possible using conventional PCR, even when working with BAL samples. Kuhnert and others (2011) noticed that suc- cessful genotyping was dependent on a suf fi ciently high concentration of M hyopneumoniae DNA in lung samples from wild boar. In this regard, the authors have recently reported the need to increase the sensitivity of some of the PCRs used for MLVA typing of this pathogen (Tamiozzo and others 2013). Sensitivity could be increased also using touchdown PCR (Korbie and Mattick 2008) and/or capillary electrophoresis (Vranckx and others 2011). This would allow M hyopneumoniae typing from minimally invasive samples (such as nasal swabs) without killing animals or performing invasive sampling (Tamiozzo and others 2013).
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Development of a Genomics Based PCR Assay for Detection of Mycoplasma pneumoniae in a Large Outbreak  in New York State

Development of a Genomics Based PCR Assay for Detection of Mycoplasma pneumoniae in a Large Outbreak in New York State

Received 20 September 2000/Returned for modification 27 November 2000/Accepted 23 January 2001 A genomics-based PCR method was developed and used to test specimens from patients involved in a large outbreak of Mycoplasma pneumoniae in a closed religious community in New York State. New P1 adhesin gene primers were designed to bind to 9 of 10 target sequences in the repetitive-element sequences obtained from the whole genome sequence of M. pneumoniae. This PCR method had a sensitivity of 0.006 CFU and a specificity of 100% for M. pneumoniae. The PCR was validated by testing a subset of patient samples by culture and comparing the results to those obtained by PCR. Of the initial 280 samples tested, 73 were positive by PCR and 22 were positive by culture. All samples positive by culture were also positive by PCR. Follow-up testing of selected patients 3 to 6 weeks after antibiotic treatment revealed that eight samples remained positive by PCR and that three samples remained positive by culture. Additionally, no nonspecific PCR inhibition was detected as a result of the specimen type, transport medium, or sample preparation methodology. The study demon- strates that the PCR described here is a rapid, sensitive, and specific method for the identification of M.
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Diagnostics of main bacterial agents of porcine respiratory diseases complex (PRDC) using PCR detection of Mycoplasma hyopneumoniae

Diagnostics of main bacterial agents of porcine respiratory diseases complex (PRDC) using PCR detection of Mycoplasma hyopneumoniae

Slovakia) was used as the positive control for the PCR detection of Mycoplasma hyopneumoniae. Cultivation To proof Pasteurella and Actinobacillus species the cultivation method on the blood agar, Gram’s stain, catalasic test and the tube test for the carbohydrate fermentation were used by Bergey’s manual (Holt et al., 1994). Important biochemical characteristics of P. multocida included positive reactions for catalase, indole and oxidase.

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Serum metabolite markers of early Mycoplasma hyopneumoniae infection in pigs

Serum metabolite markers of early Mycoplasma hyopneumoniae infection in pigs

Abstract Mycoplasma hyopneumoniae, the primary pathogenic bacterium causing enzootic pneumonia, significantly affects worldwide swine production. The infection is usually persistent and bacterial identification and isolation of M. hyo- pneumoniae in clinical samples are challenging due to the fastidious requirements for its growth. Hence, new practical surveillance tools that improve or complement existing diagnostics on M. hyopneumoniae are desirable, especially in early infection. The objective of this study was to identify potential metabolite markers of early M. hyopneumo- niae infection in pigs through metabolomics analysis. Samples obtained from pigs in a previous M. hyopneumoniae experimental infection were used in this study. Briefly, two pigs served as mock inoculated controls and ten pigs were intra-tracheally inoculated with M. hyopneumoniae. Sera, laryngeal swabs (LS), and tracheo-bronchial lavage fluid (TBLF) were collected from all pigs at 0, 2, 5, 9, 14, 21 and 28 days post-inoculation (dpi). Bronchial swabs (BS) were collected post-mortem at 28 dpi. Mycoplasma hyopneumoniae infection was confirmed by PCR in LS, TBLF and BS.
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Serum metabolite markers of early Mycoplasma hyopneumoniae infection in pigs

Serum metabolite markers of early Mycoplasma hyopneumoniae infection in pigs

Mycoplasma hyopneumoniae infection in pigs Meera Surendran Nair 1 , Dan Yao 2 , Chi Chen 2 and Maria Pieters 1* Abstract Mycoplasma hyopneumoniae, the primary pathogenic bacterium causing enzootic pneumonia, significantly affects worldwide swine production. The infection is usually persistent and bacterial identification and isolation of M. hyo- pneumoniae in clinical samples are challenging due to the fastidious requirements for its growth. Hence, new practical surveillance tools that improve or complement existing diagnostics on M. hyopneumoniae are desirable, especially in early infection. The objective of this study was to identify potential metabolite markers of early M. hyopneumo- niae infection in pigs through metabolomics analysis. Samples obtained from pigs in a previous M. hyopneumoniae experimental infection were used in this study. Briefly, two pigs served as mock inoculated controls and ten pigs were intra-tracheally inoculated with M. hyopneumoniae. Sera, laryngeal swabs (LS), and tracheo-bronchial lavage fluid (TBLF) were collected from all pigs at 0, 2, 5, 9, 14, 21 and 28 days post-inoculation (dpi). Bronchial swabs (BS) were collected post-mortem at 28 dpi. Mycoplasma hyopneumoniae infection was confirmed by PCR in LS, TBLF and BS. Serum metabolites were profiled using high-resolution liquid chromatography–mass spectrometry (LC–MS) analysis. Metabolite markers were identified by structural analysis following multivariate analysis of LC–MS data. The results showed that M. hyopneumoniae infection time-dependently altered the serum levels of selective amino acids and fatty acids. α-Aminobutyric acid and long-chain fatty acids were markedly increased at 14 and 21 dpi in inoculated pigs (p < 0.05). These results indicated that M. hyopneumoniae infection caused systemic changes in host metabolism, warranting further studies to determine underlying biochemical and physiological mechanisms responsible for the observed changes.
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A duplex real time PCR assay for the detection and quantification of avian reovirus and Mycoplasma synoviae

A duplex real time PCR assay for the detection and quantification of avian reovirus and Mycoplasma synoviae

Li Huang 1,2 , Zhixun Xie 1* , Liji Xie 1 , Xianwen Deng 1 , Zhiqin Xie 1 , Sisi Luo 1 , Jiaoling Huang 1 , Tingting Zeng 1 and Jiaxun Feng 2 Abstract Background: Infectious arthritis in broilers represents an economic and health problem, resulting in severe losses due to retarded growth and downgrading at the slaughterhouse. The most common agents associated with cases of infectious arthritis in poultry are avian reovirus (ARV) and Mycoplasma synoviae (MS). The accurate differentiation and rapid diagnosis of ARV and MS are essential prerequisites for the effective control and prevention of these avian pathogens in poultry flocks. This study thus aimed to develop and validate a duplex real-time PCR assay for the simultaneous detection and quantification of ARV and MS.
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Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations

Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations

of individual transformants in non-selective Friis medium was determined. Following a total of 15 passages in Friis medium containing no tetracycline all individual transfor- mants retained their resistance to tetracycline upon subse- quent culture in medium containing tetracycline. Linker PCR was repeated for each individual transformant, and agarose gel electrophoresis showed that the amplicons gen- erated were identical in size to those obtained prior to serial passaging. Direct DNA sequencing of PCR amplicons was performed and this confirmed that the transposon had inserted into the host cell chromosome between thymine and adenine residues as expected. Where sequencing at both ends of the transposon was possible, the transposon insertion sites were found to match. Southern analysis was performed on total DNA extracted from the same 11 trans- formants to confirm that only single transposon insertions were present. For 10 of the mutants, a single band was present using a DNA probe specific for tetM contained within the transposon (Figure 4). For mutant 1, a small in- tense band was present along with a larger feint band. Only one insertion site was identified for this transformant by linker PCR and this insertion site was confirmed by DNA sequencing. It is possible that two bands were generated by Southern analysis by a failure to adequately digest the DNA to completion with HindIII.
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Impact of diversity of Mycoplasma hyopneumoniae strains on lung lesions in slaughter pigs

Impact of diversity of Mycoplasma hyopneumoniae strains on lung lesions in slaughter pigs

showed that although M. hyopneumoniae was detected in the scalding water, the lungs of SPF pigs remained nega- tive by nested PCR [25]. The prevalence and severity of pneumonia lesions at slaughter were significantly higher in batches where more different M. hyopneumoniae strains were found, illustrat- ing for the first time the importance of strain diversity at batch level. The severity of Mycoplasma-like lesions, the prevalence of pneumonia and the prevalence of fissures was significantly higher in batches of CAT 3 compared to CAT 1, and numeric differences were obtained when batches of CAT 2 were compared to CAT 1. The effect of batch was significant in all models, indicating that there is quite some variation between successive batches in a herd. It also indicates the importance of investigating more batches from each herd.
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Multilocus Sequence Typing of Mycoplasma hyorhinis Strains Identified by a Real Time TaqMan PCR Assay

Multilocus Sequence Typing of Mycoplasma hyorhinis Strains Identified by a Real Time TaqMan PCR Assay

hyorhinis and on a collection of bacterial strains phylogenetically close to M. hyorhinis. MLST has the added advantages of providing meaningful information about population structure. The MLST work here demonstrates that sequence variation also occurs within housekeeping genes, indicating than the core genome is also variable. However, the large majority of variation was synony- mous, meaning that there was a smaller amount of variation in the expressed proteins. The large number of synonymous sub- stitutions detected suggested that most nonsilent mutations are eliminated through purifying selection. Analysis was also car- ried out to determine what the relative contributions of muta- tion and homologous recombination were in the genetic diver- sity seen among M. hyorhinis strains. It was found that diversity was probably due to recombination, with mutation playing a much smaller role. Homologous recombination was also found to be high for bacteria with walls, such as Neisseria meningitidis, and for wall-less bacteria, such as Mycoplasma hyopneumoniae and M. hominis (21, 26, 44). Due to the high frequency of recombina- tion observed in M. hyorhinis, it was surprising to observe identi- cal or nearly identical MLST genotypes (which differ by only one allele). The clonality of our M. hyorhinis population was tested by BURST analysis (45). A unique clonal complex was identified (26 strains corresponding to the ancestral group comprising ST1, which contained ATCC 25021, MCLD, and GDL-1), and only seven STs were not linked to the clonal complex and were single- tons (ST3, ST14, ST19, ST20, ST27, ST28, and ST29). Clonal groupings were also identified in Helicobacter pylori and Staphylo- coccus aureus, despite a rich history of interstrain recombination (46, 47). Søgaard et al. (44) showed that the frequency of recom- bination in M. hominis is not correlated with the level of variability and that recombination does not induce more variation in the genes but rather shuffles the existing mutations, thereby creating new alleles. Recombination in M. hyorhinis is particularly inter- esting, since no phages or plasmids have been detected in this mycoplasma, and no mechanisms that mediate DNA uptake or recombination have been described. However, Schubert et al. (48) showed that homologous DNA recombination plays a major role in horizontal transfer of genes within the species E. coli, and Sirand-Pugnet et al. (49) demonstrated that a significant number of genes underwent horizontal transfer among different myco- plasma species that share the same ruminant hosts.
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A comparison study between GeXP based multiplex PCR and serology assay for Mycoplasma pneumoniae detection in children with community acquired pneumonia

A comparison study between GeXP based multiplex PCR and serology assay for Mycoplasma pneumoniae detection in children with community acquired pneumonia

laboratory diagnosis methods of M. pneumoniae infec- tion for childhood CAP in larger clinical database has not been reported. In the present study, we applied a multiplex-PCR, named GeXP assay, to detected 13 types of pathogens including M. pneumoniae in 3146 sputum samples from hospitalized CAP children. Meanwhile, their serum specimens were also collected to be tested the antibody against M. pneumoniae. After the compari- son between serology and GeXP assays on the same pa- tient, about 93.7% of cases were diagnosed to be either positive or negative based on these 2 methods. We ran- domly chose 46 samples from 198 samples that had been tested to be inconsistent by serology and GeXP assays, redo the Mp-SAT and found 97.8% diagnosis agreement between SAT and GeXP assays, but not with the ser- ology testing. Further analysis revealed that children in whom M. pneumoniae was only examined to be positive by GeXP or serology were significantly younger than those of both methods positive. About 37% of patients have virus-Mp coinfection, which were most observed in infantile patients. Seasonal distribution analysis revealed that the virus-Mp coinfection were predominant in spring and winter, while Mp-only infection was more frequently identified in summer and autumn.
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Rapid Detection of Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum Organisms in Genitourinary Samples by PCR Microtiter Plate Hybridization Assay

Rapid Detection of Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum Organisms in Genitourinary Samples by PCR Microtiter Plate Hybridization Assay

of Urology, Gifu University School of Medicine, Gifu City, Gifu 500-8705, 3 Japan Received 3 July 2002/Returned for modification 13 August 2002/Accepted 23 February 2003 We present a method for detecting the presence of Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum organisms, which are thought to be associated with nongonococcal urethritis (NGU) and other genitourinary infections, in clinical samples. This method consists of PCR amplification of a part of the 16S rRNA gene followed by 96-well microtiter plate hybridization assay using four species-specific capture probes to detect the targets. To test the efficacy of this method, we applied it to the detection of the four species in the urine of patients with NGU. There were no cross-reactions with other human mycoplasmas or ureaplasmas, and the PCR-microtiter plate hybridization assay detected as few as 10 copies of the 16S rRNA gene of each of the four species. Based on these results, this PCR-microtiter plate hybrid- ization assay can be considered an effective tool for the diagnosis of genitourinary infections with mycoplasmas or ureaplasmas.
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