Top PDF PCR-based assay for Mycoplasma hyopneumoniae

PCR based assay for Mycoplasma hyopneumoniae

PCR-based assay for Mycoplasma hyopneumoniae

The kit of claim 3 wherein said probe is an oligonucle otide consisting of the nucleotide sequence product of each oligonucleotide primer that is comple 20 mentary strand; and at an eife[r]

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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

Patients were asked to cough and the expectorated spu- tum was collected. If the child is too young to cough, a sterile negative pressure suction catheter is applied to obtain the oropharyngeal suction (OPS) into transport tube containing 1 ml DMEM medium with 2% heat- inactivated fetal calf serum, 50 IU/ml of penicillin and 100 μg/ml of streptomycin (Gibco, Beijing, China). The sample was stored at 4 °C for the same day pathogen nu- cleic acid extraction. Total nucleic acid (both DNA and RNA) was extracted from 200 μL sputum sample and eluted into 30 μL nuclease-free water by the EasyPure Viral DNA/RNA Kit (QSJBio, Beijing, China) in accord- ance with the manufacturer’s instructions. Afterwards, 3 μL of extracted nucleic acid was analyzed with the GeXP-based assay. The remaining sputum samples were added into equal volume of preservative solution and stored at a − 80 °C freezer.
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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

An additional question addressed in the present outbreak study was what effect treatment with the antibiotic azithromy- cin would have on the ability of members of the community to harbor M. pneumoniae. To address this question, secondary specimens were taken from selected members of the commu- nity 3 to 6 weeks following the initial specimen collection from a group of 53 patient specimens that were initially positive by PCR, 8 specimens remained positive by PCR (including three specimens that remained culture positive). Because this group of specimens contained specimens from both symptomatic and asymptomatic patients, no clear link between the duration of therapy or the presentation of the infected individual and the persistence of the organism can be concluded from these re- sults. However, a marked decrease in the number of affected individuals was found after the 3- to 6-week period. Azithro- mycin therapy has previously been found to be effective at preventing outbreaks of infection with M. pneumoniae (9). Additionally, the combined use of the 1,500-mg cumulative dose of azithromycin and standard epidemic control measures has been associated with a significant reduction in the rate of transmission of M. pneumoniae (13).
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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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A field evaluation of two vaccines against Mycoplasma hyopneumoniae infection in pigs

A field evaluation of two vaccines against Mycoplasma hyopneumoniae infection in pigs

Herd A was located on a small island close to Funen. It was a specific pathogen free (SPF) herd but infected with M. hyopneumoniae, Porcine reproductive and re- spiratory syndrome virus (PRRS) types 1 and 2 and hous- ing only nursery and grower/finisher pigs. The herd received 575 four-week-old piglets every 4th weeks, which were assigned to two sections. The two sections of nursery pigs were commingled in one grower/finisher section. Both nursery and grower/finisher units were operated on an all-in/all-out basis by section. The pigs were treated for oedema disease with antibiotics during the first three weeks after arrival. Before inclusion in the trial, lung lesion scores were evaluated in 30 pigs from one delivery batch as described by [2]. This evaluation found that 87% of the pigs had catarrhal bronchopneumonia. M. hyopneumoniae was detected by an in-house polymerase chain reaction (PCR) assay at the Danish Veterinary Institute (Copenhagen, Denmark) from one lung with catarrhal bronchopneumonia, and antibodies against M. hyopneumoniae were detected by enzyme-linked immunosorbent assay (ELISA) at the Danish Veterinary Institute in ten out of ten finishers [7].
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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

M ycoplasmas are commonly described as the simplest and smallest self-replicating bacteria because of their total lack of a cell wall, the paucity of their metabolic pathways, and the small size of their genome (1). While mycoplasmas are considered the simplest free-living organisms, several species are successful pathogens of humans and animals. Mycoplasma hyorhinis, a major contaminant of mammalian tissue cultures in laboratories world- wide, usually infects pigs, leading to respiratory tract disease and inflammation of the chest and joints (2). Polyserositis, arthritis, pneumonia, otitis, and conjunctivitis are clinical disorders associ- ated with M. hyorhinis infection (2). In the porcine respiratory disease complex, M. hyorhinis appears to be frequently associated with Mycoplasma hyopneumoniae, the primary agent of enzootic pneumonia (3). Respiratory diseases remain the most challenging health problems in pig production worldwide (4). For example, in France, a prevalence study showed that 72.4% of slaughter pigs suffered from pneumonia and that 14.4% had pleuritis (5). These lung diseases result in financial losses due to poor growth perfor- mance, reduced feeding efficiency, and higher medication costs and have an adverse effect on pig welfare (4). Moreover, the ad- ministration of antimicrobials may have a potential negative effect on human health when associated with food-borne contamina- tion by resistant pathogens (even if M. hyorhinis is generally sus- ceptible to the antibiotics used against M. hyopneumoniae) or re- sistant commensal bacteria (6).
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Mycoplasma hyopneumoniae Potentiation of Porcine Reproductive and Respiratory Syndrome Virus Induced Pneumonia

Mycoplasma hyopneumoniae Potentiation of Porcine Reproductive and Respiratory Syndrome Virus Induced Pneumonia

Pathologic examination. Pigs were necropsied at either day 3, day 10, or day 28, as outlined in Table 1. The right rib cage was reflected, and the lungs were removed and evaluated for macroscopic lesions. A portion of lung was aseptically collected for M. hyopneumoniae and PRRSV isolation, fluorescent antibody assay (FA), immunohistochemistry (IHC), and histopathologic examination. The lungs were then lavaged, and BAL fluid was obtained. Lesions consistent with mycoplasmal pneumonia (dark-red-to-purple consolidated areas) were sketched on a standard lung diagram. The proportion of lung surface with lesions was determined from the diagram by using a Zeiss SEM-IPS image analyzing system (23). In contrast to mycoplasma-induced lesions, PRRSV-infected lungs were characterized by parenchyma that was mottled tan and rubbery and failed to collapse. The lung lesions were scored by using a previously developed system based on the approximate volume that each lobe contributes to the entire lung: the right cranial lobe, right middle lobe, cranial part of the left cranial lobe, and caudal part of the left cranial lobe each contribute 10% each of the total lung volume, the accessory lobe contributes 5%, and the right and left caudal lobes each contribute 27.5% (13). These scores were then used to calculate the total lung lesion score based on the relative contribution of each lobe.
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Individual risk factors for Mycoplasma hyopneumoniae infections in suckling pigs at the age of weaning

Individual risk factors for Mycoplasma hyopneumoniae infections in suckling pigs at the age of weaning

DNA isolation from nasal swabs was always conducted at the day of sampling. The top of each swab was clipped and incubated in 1.5 ml sterilized Tris-EDTA buffer for 30 min at 56°C. After transferring the top of the swab into a shortened filter tip, which was placed in a new re- action tube, this tube was centrifuged at 18,000 g for 15 sec. Subsequently, the shortened filter tip containing the swab was discarded, while the liquid on the bottom of the reaction tube was transferred into the correspond- ing reaction tube, which contained the Tris-EDTA buffer from the first incubation. The samples were centrifuged at 18,000 g lasting 20 min. After discarding the liquid, pellets were submitted to DNA isolation using a silica- membrane-based spin kit according to the manufac- turer’s instructions (QIAamp DNA Mini kit, Qiagen). Amplification of DNA was performed using a multiplex real-time PCR [16,17] on an AB 7500 system (Life- technologies).
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Efficacy of three innovative bacterin vaccines against experimental infection with Mycoplasma hyopneumoniae

Efficacy of three innovative bacterin vaccines against experimental infection with Mycoplasma hyopneumoniae

The study was performed after approval by the Ethi- cal Committee for Animal Experiments of the Faculty of Veterinary Medicine, Ghent University (approval number EC2017/120). Fifty-three M. hyopneumo- niae-free Rattlerlow-Seghers piglets (RA-SE Genet- ics NV, Ooigem, Belgium) were enrolled in the study. All animals originated from a herd that has been M. hyopneumoniae-free for many years based on repeated serological testing, nested PCR testing on tracheo- bronchial swabs, and absence of clinical signs and pneumonia lesions at slaughter. The piglets arrived at the experimental facilities of the Faculty of Veterinary Medicine, Ghent University, Belgium at 32 days of age. They were housed in stables with absolute air filters for impending particles (HEPA U15) on both incoming and outgoing ventilation shafts and fed ad libitum with a non-antimicrobial-supplemented diet. On the day of arrival (D-7), the piglets were randomly allocated into three vaccination groups and a PBS-injected control group (PCG) of 12 piglets each. Five pigs were included as a non-challenge control group (NCG). After an accli- matization period of 1 week, the pigs of the vaccination groups were prime-boost vaccinated intramuscularly (IM) at day (D) 0 and D14 of the study with 2  mL of experimental bacterin. The pigs of the PCG and NCG received 2 mL sterile PBS IM at both vaccination days. The pigs of the vaccinated groups and the PCG were experimentally infected by endotracheal inoculation of the highly virulent M. hyopneumoniae strain F7.2C (7  mL culture medium containing 10 7 CCU/mL) on
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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

Hybridization. Amplified products of M. genitalium, M. hominis, U. parvum, and U. urealyticum were detected by hybridization-based microtiter plate assay with species-specific oligonucleotide probes. We designed the capture probes in the species-specific regions demonstrated by the alignment of human mycoplas- mas and ureaplasmas (GenBank accession numbers AB069810 to AB069824). Four capture probes—Mgen-P3-Am (5⬘-TCGGAGCGATCCCTTCGGT-3⬘) specific for M. genitalium, Mhom-P10-Am (5⬘-GACACTAGCAAACTAGAGT TAG-3⬘) specific for M. hominis, Upar-P6-Am (5⬘-GTCTGCCTGAATGGGTC GGT-3⬘) specific for U. parvum, and Uure-P4-Am (5⬘-GGCTCGAACGAGTC GGTGT-3⬘) specific for U. urealyticum—were 5⬘ aminated with a six-carbon spacer. We also designed a capture probe to detect the amplified product of the internal control (IC-P4-Am: 5⬘-CTAGCTGTCGGCTGGAATTC-3⬘). These probes were each immobilized to a microtiter plate, DNA-BIND 1 ⫻ 8 strip well plate (Corning Inc., Corning, N.Y.), as described in the manufacturer’s instruc- tions. The amplified products were denatured by boiling for 5 min and were quickly chilled in ice. One hundred microliters of hybridization buffer (5⫻ SSC [1⫻ SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.02% sodium dodecyl sulfate) was added to the wells of each microtiter plate immobilized by the capture probes. Five microliters of denatured amplicons was then added, and hybridization was carried out at 37°C for 90 min. Following hybridization, the wells were washed twice (soaking for 5 min) with wash buffer I (360 ␮l/well; 0.2⫻ SSC, 0.1% sodium dodecyl sulfate). One hundred microliters of streptavidin- peroxidase (Roche Molecular Biochemicals) was added per well and allowed to incubate for 15 min at 37°C. Then the wells were washed twice (soaking for 5 min) with wash buffer II (360 ␮l/well; 0.1% Tween 20 in phosphate-buffered saline). One hundred microliters of BM Blue POD Substrate (Roche Molecular Biochemicals) was added per well and allowed to sit for 10 min at room tem- perature. The reaction was stopped by the addition of hydrosulfuric acid (100 ␮l/well), and the OD at 450 nm was measured by the Novapath Microplate Reader (Bio-Rad, Richmond, Calif.). The cutoff OD value was determined to be 0.300 by measuring the negative control value 20 times. The mean value plus 4
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Herd specific risk factors for Mycoplasma hyopneumoniae infections in suckling pigs at the age of weaning

Herd specific risk factors for Mycoplasma hyopneumoniae infections in suckling pigs at the age of weaning

Subsequently to sampling, the nasal swabs were directly submitted to DNA isolation. The top of each swab was clipped and transferred into a reaction tube containing 1.5 mL Tris-EDTA (TE) buffer. After 30 min of incuba- tion at 56ºC, the top of the swab was transferred into a shortened filter tip, which was placed in a new reaction tube, whereas the liquid in the primary tube was stored. The construct in the secondary tube was centrifuged at 18,000 g lasting 15 sec. Subsequently, the filter tip containing the top of the swab was discarded and the liquids of both reaction tubes were merged and mixed in a new one. After centrifugation at 18,000 g lasting 20 min, pellets were submitted to DNA isolation using a silica-membrane-based spin kit according to the manu- facturer’s instructions (QIAamp DNA Mini kit, Qiagen). A swab previously dipped into TE buffer containing a modified Escherichia coli (K12 p_MYO_REP & p_MYO_ABC) served as an external positive control for successful DNA isolation and amplification in every test setup. The genetically modified strain of E. coli is carry- ing plasmids, which had been extended by the PCR target sequences of the REP and ABC genes [13]. Swabs dipped in pure TE buffer were used as negative control. The DNA was examined using real-time PCR amplifying sequences of the REP- and ABC-genes of M. hyopneumoniae [13]. The assay had been modified to a
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Prospective Evaluation of ResistancePlus MG, a New Multiplex Quantitative PCR Assay for Detection of Mycoplasma genitalium and Macrolide Resistance

Prospective Evaluation of ResistancePlus MG, a New Multiplex Quantitative PCR Assay for Detection of Mycoplasma genitalium and Macrolide Resistance

and assessment of macrolide resistance. Patients infected with M. genitalium 23S rRNA mutants are predicted to fail treatment. This study aimed to evaluate the assay with consecutive samples sent to the laboratory and included a mixture of specimens from symptomatic and asymptomatic patients that had been sent to the laboratory for testing. Although the details of the individual patient symptoms were not available, the alarmingly higher prevalence of M. genitalium and resistance mutations detected in the male population, which consisted primarily of men from a sexual health clinic who were symptomatic with urethritis, highlights the importance of utilizing this assay for detec- tion of M. genitalium and resistance testing to inform management of disease. This assay offers a considerable advantage for the rapid detection of macrolide-resistant strains and can be incorporated into diagnostic algorithms which can individualize antimicrobial therapy to ensure rapid delivery of agents to which the organism is susceptible. Where azithromycin is used as the first-line therapy, simultaneous avail- ability of M. genitalium detection and detection of macrolide resistance mutations would allow clinicians to rapidly recall patients to provide a more appropriate second- line treatment in comparison to waiting for up to 4 weeks for test of cure or treatment failure. Where doxycycline rather than azithromycin is used as first-line therapy for sexually transmitted infection (STI) syndromes, patients with M. genitalium infection can be recalled for treatment with an antimicrobial based on the macrolide resistance profile. Macrolide-susceptible strains can be treated with azithromycin and macrolide- resistant strains with a quinolone such as moxifloxacin. Future assays that provide quinolone resistance data would assist in further refining clinical algorithms by iden- tifying patients with dual-class resistance. Of note, 9% of M. genitalium strains at Melbourne Sexual Health Centre in Melbourne, Australia, currently have dual-class resistance (macrolide and quinolone), resulting in the need to use antimicrobials such as pristinamycin which have very limited availability outside Europe (25). Clinical algorithms such as this that incorporate resistance testing and individualize care have the potential to greatly improve microbial cure, to promote antimicrobial stewardship, and to reduce clinic visits and, ultimately, the spread of antibiotic-resistant bacteria in populations.
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Four color multiplex real time PCR assay prototype targeting azithromycin resistance mutations in Mycoplasma genitalium

Four color multiplex real time PCR assay prototype targeting azithromycin resistance mutations in Mycoplasma genitalium

The specificity of the M + IC multiplex (2058 mutants MG (FAM dye), 2059 mutants MG (YY dye), total MG (Mg219 gene, ROX dye) and IC (Cy5 dye)) for MG DNA was tested against commercial genomic DNA of the fol- lowing bacterial, yeast and viral species: Chlamydia tra- chomatis, Chlamydophila pneumoniae, Clostridium difficile, Escherichia coli, Gardnerella vaginalis, Lactoba- cillus acidophilus, Lactobacillus brevis, Lactobacillus jen- senii, Lactobacillus reuteri, Mycoplasma arginini, Mycoplasma pneumoniae, Mycoplasma hominis, Myco- plasma orale, Mycoplasma salivarium, Neisseria gonor- rheae, Neisseria meningitidis serogroup A, Neisseria meningitidis Serogroup B, Neisseria meningitidis FAM18 serogroup C, Neisseria meningitidis M1883 serogroup C, Trichomonas vaginalis, Ureaplasma parvum, Urea- plasma urealyticum, Candida albicans, Candida glab- rata, Herpesvirus 1 (HSV-1), Herpesvirus 2 (HSV-2) and Herpesvirus 5 (CMV). These species were selected based either on their probability of being present in clinical samples tested for MG (AZM-resistant or not) or on their phylogenetic proximity to MG. All wells showed a positive signal for the UIC, validating the PCR results. No signal for any target was obtained with any of these non-MG species, while the positive control was positive for each target.
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Use of trachea-bronchial swab qPCR testing to confirm Mycoplasma hyopneumoniae seropositivity in an SPF breeding herd

Use of trachea-bronchial swab qPCR testing to confirm Mycoplasma hyopneumoniae seropositivity in an SPF breeding herd

breeding companies that currently deliver high health breeding animals to their customers. Serology using ELISA is the preferential approach in order to screen for M. hyopneumoniae [9–14]. In case of positive serology, further decisions on farm health status and the related consequences should be based on additional confirm- ation tests. Clinical diagnosis of enzootic pneumonia can be verified by serological analysis [10]. However, in SPF programmes, the herd prevalence of M. hyopneumoniae infections is often low and the positive herd predict- ive value of a serological result decreases progres- sively with the decreasing herd prevalence [15]. Moreover, ELISA testing of sera from naturally in- fected pigs does not detect early-stage infection prior to seroconversion [16, 17], and infection and vaccin- ation responses are indistinguishable. Under field con- ditions, the mean time to onset of coughing following an M. hyopneumoniae infection was 13 days, whereas the mean time between onset of coughing and seroconversion as measured by ELISA was 9 days [10]. Recent research has shown that currently used ELISA tests only start showing a seroconversion from 21 days post-infection on- wards [18]. The percentage of animals seroconverting in the early stages of M. hyopneumoniae infection using one of the commercially available M. hyopneumoniae ELISAs remains relatively low (16–22% at 21 days and 35–45% at 28 days post-infection) [18]. This implies that a large number of samples is needed to reliably detect the pres- ence of M. hyopneumoniae within the monitored herd. In the Danish SPF program, the final verification of herd in- fection with M. hyopneumoniae is consequently per- formed by demonstration of the agent [10]. A recent comparative study on diagnostic sampling approach for M. hyopneumoniae detection showed that laryngeal swabs were a reliable option to establish early detection of M. hyopneumoniae, followed by brocho-alveolar lavage fluids and nasal swabs [18]. Other innovative sampling tech- niques, such as tracheo-bronchial swab (TBS) sampling [6, 7, 19] have been introduced in combination with PCR detection of M. hyopneumoniae to reliably detect the pathogen of infected animals. The objective of the current case report is to show that TBS sampling is a suitable method to confirm a suspect M. hyopneumoniae-seroposi- tive situation.
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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

The genome of Mycoplasma hyopneumoniae encodes several immunodominant proteins, including a cytosolic protein (p36), three membranous proteins (p46, p65, and p74), and an adhesin (p97). Cross-reactions with M. 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. hyopneumoniae infection, and no false-positive results were detected when 62 apparently normal lungs were tested. On the other hand, with the single p46-PCR method a sensitivity of 86.6% (26 of 30) and a specificity of 96.7% (60 of 62) were obtained in comparison with the necropsy findings. A mixed infection with M. hyorhinis was diagnosed in 13.3% (4 of 30) of the cases by using species-specific primers for the heterologous p37 gene. The sensitivity of the single p36-PCR method for the detection of M. hyopneumoniae, when tested on tracheo- bronchial swabs, was 100% (20 positive samples), with a specificity of 93.3% (14 of 15 negative samples), compared to the necropsy findings. Both expected amplicons were obtained with 86.6% (26 of 30) positive lungs when p36 and p46 primers were used simultaneously (multiplex PCR) to further increase the specificity of the PCR assay.
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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

hyopneumoniae (Mhp165 seqF and Mhp165 seqR) (Table 3). A panel of forward and reverse primers was then designed (Mhp165 F g-a, Mhp165 R t-c, Mhp165 R t-c g-a, and Mhp165 R g-a) (Table 3) based on the sequence results (Fig. 1). The primers were then tested for their abilities to detect the isolates possessing single-nucleotide polymorphisms (SNPs). One set of for- ward and reverse primers (Mhp165 F and Mhp165 R) (Table 3) was selected and shown to be unique to M. hyopneumoniae by BLAST analysis. This set was further tested for specificity by use of a SYBR green assay performed as described by the manufacturer (QuantiTect SYBR green PCR kit; Qiagen) with DNA from M. hyopneumoniae, M. flocculare, M. hyorhinis, and M. hyo- synoviae. All real-time assays were performed on a Rotor-Gene RG-3000 (Corbett Research, San Francisco, CA). A checkerboard assay of primers at 100 nM, 300 nM, and 900 nM versus probe concentrations of 50 nM, 150 nM, and 250 nM was used to optimize the real-time protocol by use of the universal PCR master mix (Applied Biosystems, Foster City, CA) according to the manufacturer’s directions. The following cycling parameters were used: 50°C for 2 min; 95°C for 10 min; and 40 cycles of 95°C, 15 s, and then 60°C for 1 min. Template DNA from four different isolates of M. hyopneumoniae was utilized in replicates of the checkerboard assay. The detection limit of the finalized assay was tested using a dilution series (10 ng/␮l to 1 fg/␮l) of chromosomal DNA from M. hyopneumoniae strain 232. The assay was also compared to a nested PCR targeting the mhp165 gene that includes an inner set of primers (8) corresponding to a previously published outer set (19) (Mhp165 outerF, Mhp165 outerR; Mhp165 innerF, Mhp165 innerR) (Table 3). By use of the GeneAmp PCR core kit (Applied Biosystems), the following reaction mixture was employed for both the inner and outer assays: 1⫻ PCR buffer II, 2.5 mM MgCl 2 , 100 ␮M each deoxynucleoside triphosphate, 0.4 mM
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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

detect down to two copies per reaction tube. However, due to the Poisson distribution of targets in dilute specimens, the realistic limit of detection was ⱖ 5 copies per assay. The accu- racy of this detection limit estimate was documented by the intra-assay reproducibility test, where nine replicates of 1 copy/ ␮ l were evaluated. Of these replicates, 33% were neg- ative. If the DNA is assumed to be randomly distributed in the solution and to follow Poisson statistics, then the PCR is ex- pected to miss exp( ⫺ 2) ⫻ 100% ⫽ 13.5% positives when an average of two DNA copies is added to the tube (correspond- ing to the concentration of 1 copy/ ␮ l). The difference between the expected and observed number of false negatives can be explained by pipetting errors (especially for low DNA concen- trations), too few replicates, or a small overestimation of the DNA concentration in the M. genitalium genomic DNA stan- dard solution. Other real-time PCR methods have been devel- oped for the detection of M. genitalium in urine samples (6, 29). The limits of detection of the TaqMan-based assay by Yoshida et al. (29) and the LightCycler PCR assay by Dupin et al. (6) were similar to the sensitivity of our LightCycler PCR, with detection limits of 10 and 5 copies per reaction tube, respectively. However, since no external reference standard exists, only a direct comparison using a blinded panel of several DNA specimens could indicate true differences in the detec- tion limits between assays. Furthermore, the more relevant performance estimate given by sensitivity and specificity can only be provided with genuine clinical specimens. To address this issue, we applied two independent real-time PCR assays targeting two different M. genitalium genes. To our knowledge, this is the first comparison between two real-time methods for the detection of M. genitalium. We found an excellent 1:1 relationship between the two PCR methods, as the slope of the regression line was very close to 1 (0.99), indicating that the quantity determined by the two assays was comparable. On average, the TaqMan assay estimated the level of M. genitalium DNA to be twofold higher than was estimated by the Light- Cycler assay. This was determined from the intercept of the regression line (Fig. 4). However, since this factor was constant over the detected range of M. genitalium DNA loads, it could probably be traced back to different standards used for deter- mination of the standard curve and should not impede the detection of relative changes measured within each assay.
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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

pathogen-free swine experimentally inoculated with M. hyopneumoniae. DNA from 11 other mycoplasma species and 17 cell-walled bacterial species colonizing the respiratory tracts of pigs was not amplified. In a field study BALFs from 40 pigs from farms with a history of chronic pneumonia were tested for M. hyopneumoniae by cultivation and by PCR (i) with BALFs incubated in Friis medium and (ii) with DNA extracted from the BALFs. In addition, PCR was performed with postmortem lung washings from 19 of the 40 pigs, and immunofluores- cence tests were carried out with sections of lungs from 18 of the 40 pigs. M. hyopneumoniae could not be detected in 18 of the 40 pigs by any of the five methods tested. The remaining 22 pigs showed a positive reaction by the PCR with DNA extracted from the BALFs and variable positive reactions by the other tests. A complete correspondence could be observed between the immunofluorescence test result and the result of PCR with DNA. The investigation shows that the PCR with DNA extracted from BALFs is a suitable technique for the sensitive and specific in vivo detection of M. hyopneumoniae.
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Comparison of Transcription Mediated Amplification and PCR Assay Results for Various Genital Specimen Types for Detection of Mycoplasma genitalium

Comparison of Transcription Mediated Amplification and PCR Assay Results for Various Genital Specimen Types for Detection of Mycoplasma genitalium

The differential detection of M. genitalium by specimen type may reflect differences in the bacterial load between vaginal swab, cervical swab, and urine specimens. Although we do not know the true preferred site of infection or the relative density of M. genitalium at different genital sites in women, Blaylock et al. (4) determined that M. genitalium is associated with vaginal epithelial cells and resides intracellularly, supporting coloniza- tion at this site. Several studies have shown an association of M. genitalium with cervicitis (1, 25, 30, 39) and urethritis in women (1), yet the interaction of M. genitalium at these sites, as well as the bacterial loads among women with and without these syndromes, has not yet been determined. Other factors specific to the specimen type might affect the sensitivity of detection. For example, to collect cervical swab specimens, clinicians typically remove the overlying mucus before collect- ing the underlying epithelium for testing by PCR. This proce- dure was originally designed for optimal culture of C. tracho- matis but has an unknown effect on the detection of M. genitalium. In addition, in many studies multiple cervical swab specimens are collected from each patient, likely reducing the amount of sample and, possibly, the sensitivity of detection per specimen. In our study, the cervix was sampled for the detec- tion of M. genitalium after specimens were collected for testing for patient management, including testing by Gram stain, cul- ture, and/or NAAT for N. gonorrhoeae and C. trachomatis. These multiple samplings may have limited the quantity of the material for M. genitalium detection and thus the sensitivity of M. genitalium detection in cervical specimens. Factors affecting the sensitivity of detection in urine specimens include the time since last urination and the patient’s ability to collect the first- void urine specimen (the first 30 ml). The detection of M. genitalium in female urine specimens may be dependent upon contamination with vaginal or cervical secretions or may be enhanced by urethral infection in a subset of women (1), which may increase the organism load and therefore the ability to detect the organism.
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Interaction between Mycoplasma hyopneumoniae and  Swine Influenza Virus

Interaction between Mycoplasma hyopneumoniae and Swine Influenza Virus

Porcine respiratory disease complex (PRDC) is an econom- ically significant respiratory disorder characterized by slow growth, decreased feed efficiency, lethargy, anorexia, fever, cough, and dyspnea (5). Diagnostic laboratories have isolated multiple pathogens from cases of PRDC, including porcine reproductive and respiratory syndrome virus (PRRSV), Myco- plasma hyopneumoniae, swine influenza virus (SIV), Actinoba- cillus pleuropneumoniae, and pseudorabies virus (PRV) (5). Of these pathogens, PRRSV, M. hyopneumoniae, and SIV are most frequently detected in 10- to 22-week-old pigs with clin- ical signs of PRDC (5). Recent studies found that infection with M. hyopneumoniae potentiated and prolonged PRRSV- induced pneumonia based on clinical, macroscopic, and micro- scopic findings (12). Van Reeth et al. found that the clinical effects of PRRSV were exacerbated with concurrent infection with SIV (19). The purpose of the study reported here was to investigate the interaction between M. hyopneumoniae and SIV.
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