Mycoplasma detection by PCR analysis

Summary The polymerase chain reaction (PCR) method was used to detect mycoplasma contamination in a panel of 42 continuous cell lines. According to the microbiological cultivation assay on agar, 29 cell lines were chronically infected and 13 cell lines were negative. Sets of outer and inner primers (nested double-step PCR) were applied which anneal to DNA sequences coding for conserved regions of the 16S rRNA. These oligonucleotides allow for the amplification of DNA regions found in at least 25 mycoplasma species (including the ones most commonly found in cell cultures), but do not cross-hybridize with DNA from eukaryotic cells. Mycoplasma-positive cell lines showed distinctive bands in ethidium bromide-stained gels, both after the first round of amplification as well as after the second PCR; all agar-negative cell lines were also unambiguously negative in the PCR assay. Thus, neither false-positive nor false-negative results occurred. Provided that the proper PCR working conditions are scrupulously observed, the PCR amplification has several outstanding advantages: high sensitivity, specificity, reliability, objectivity, speed, and simplicity.     

A real-time PCR assay for detection and quantification of Mycoplasma agalactiae DNA

AIMS: The aim of this study was to develop a rapid, sensitive, specific tool for detection and quantification of Mycoplasma agalactiae DNA in sheep milk samples. METHODS AND RESULTS: A real-time polymerase chain reaction (PCR) assay targeting the membrane-protein 81 gene of M. agalactiae was developed. The assay specifically detected M. agalactiae DNA without cross-amplification of other mycoplasmas and common pathogens of small ruminants. The method was reproducible and highly sensitive, providing precise quantification of M. agalactiae DNA over a range of nine orders of magnitude. Compared with an established PCR assay, the real-time PCR was one-log more sensitive, detecting as few as 10(1) DNA copies per 10 microl of plasmid template and 6.5x10(0) colour changing units of reference strain Ba/2. CONCLUSIONS: The real-time PCR assay is a reliable method for the detection and quantification of M. agalactiae DNA in sheep milk samples. The assay is more sensitive than gel-based PCR protocols and provides quantification of the M. agalactiae DNA contained in milk samples. The assay is also quicker than traditional culture methods (2-3 h compared with at least 1 week). SIGNIFICANCE AND IMPACT OF THE STUDY: The established real-time PCR assay will help study the patterns of shedding of M. agalactiae in milk, aiding pathogenesis and vaccine efficacy studies.     

A quantitative TaqMan PCR assay for the detection of Mycoplasma suis

Aim: To develop a TaqMan probe‐based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. Methods and Results: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay’s specificity, detection limit, intra‐ and inter‐assay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100‐fold more sensitive than the cPCR. No cross‐reactivity with nontarget pig mycoplasmas was observed. An average of 1·62 × 10¹¹ and 2·75 × 10⁸ target copies ml^?1 of blood were detected in the acutely and chronically infected pigs, respectively. Three (7·5%) pigs and 32 (80·0%) sows were positive while all peccaries were negative for Myc. suis. Conclusion: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. Significance and Impact of the Study: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.     

Multiplex real-time PCR for the simultaneous detection of herpes simplex virus, human herpesvirus 6, and human herpesvirus 7

A simultaneous detection system to quantify HSV, HHV-6, and HHV-7 DNA via multiplex real-time PCR using different fluorochromes was developed. The minimum quantitative level established via this multiplex assay was four copies per reaction for HSV type 1, four copies for HHV-6, and three copies for HHV-7, respectively. The dynamic range encompassed at least six orders of magnitude. The system was specific and reproducible even in the presence of large amounts of other viral DNA. We then applied this multiplex real-time PCR assay to 105 CSF specimens obtained from subjects less than 15 years old in whom a diagnosis of viral encephalitis/encephalopathy was suspected on clinical grounds. The detection rate for each viral DNA was 6.7% for HSV, 9.5% for HHV-6, and 1.9% for HHV-7. These results indicate that our system is reliable and may be useful for the rapid diagnosis of viral encephalitis/encephalopathy.     

Use of PCR methods for detection of selected genes of Mycoplasma pneumoniae

The aim of this study was standardization of PCR for the detection of gene encoding the P1 protein, 16S rRNA and elongation factor Tu of M. pneumoniae. A total of 13 strains of M. pneumoniae, 28 strains of other mycoplasmas and 14 strains of different bacteria causing respiratory tract infections were tested. In all of tested M. pneumoniae strains the presence of the sought genes was confirmed. The specificity of DNA was confirmed by the restriction endonuclease analysis with enzymes Hind III, Alu I and Hha I. With none of primers specific for the M. pneumoniae genes amplification of DNA from other bacteria was noted. The PCR method with the selected primers allowed to detect from 10(2) to 10(4) cfu M. pneumoniae/ml suspended in broth. The obtained results indicate that the PCR method can be used for detection of M. pneumoniae genes. A very good sensitivity and specificity predestine++ PCR as a potential quick diagnostic method for identification of M. pneumoniae in clinical specimens.     

Modification and optimization of PCR methods for detection of MIE region from human herpesvirus 5

Human herpesvirus 5 (HHV-5, formerly known as CMV) is a beta-herpesvirus widely spread within a population. Thus, HHV-5 infections are a serious matter of concern in a group of immunocompromised patients. The goal of the study was modification and optimization of conventional PCR method developed for the detection of HHV-5 DNA to the real-time variant (RTmPCR) and determination of analytical resolution of the modified methods. Thirty plasma samples were tested for the presence of HHV-5 DNA using the LightCycler system with two different methods--one with SYBR Green I fluorochrome method and second one using TaqMan fluorescent probes and a qualitative in-house gel-stained PCR assay using primers that amplify part of HHV-5 MIE gene. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of HHV-5 DNA in range between 10(0) and 10(-6). For comparison typical end-point detected PCR for cytomegalovirus detection with the same DNA dilutions was made. The sensitivity of novel method was about 100-fold higher than older one. Both LightCycler assays detected HHV-5 DNA in 27 samples, also which were negative by the gel-stained PCR. Analysis of the available clinical and serological data associated with these samples suggested that the real-time results in all of these cases were true positive. The conclusion is that real-time PCR methods are more sensitive than the conventional PCR used in this study. The additional sensitivity was valuable for detection of patients with low-copy viremia. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler instrument are favorable for the use of this system in the detection of HHV-5 DNA in clinical specimens.     

First LightCycler real-time PCR assay for the quantitative detection of Mycoplasma suis in clinical samples

Mycoplasma suis cannot be cultivated in vitro. Therefore, PCR-based methods are irreplaceable for the diagnosis of M. suis infections especially when clinical symptoms are not evident. Currently, no easy and reliable method allowing the quantitative detection of M. suis is available. This report describes the development of a quantitative LightCycler PCR assay based on the msg1 gene of M. suis (LC MSG1 PCR). No PCR signals were obtained with closely related haemotrophic and non-haemotrophic mycoplasmas, with other bacteria, and with M. suis-free blood and tissue arguing for a high analytical specificity. Test sensitivity was found to be 100%, and test specificity 96.7%. To test the diagnostic suitability of the LC MSG1 PCR, 25 pigs with clinical porcine eperythrozoonosis and 25 healthy pigs were investigated. All ill pigs revealed a positive real-time PCR result whereas only one healthy pig was detected to be M. suis-infected. M. suis was quantitatively detected in 19 blood specimens of 100 sows from Switzerland and in 17 of 160 post-weaning piglets from Germany. In conclusion, this new LC MSG1 PCR assay represents a powerful tool for the improvement of the current M. suis diagnosis and for prevalence and pathogenesis studies.     

Development of a rotor-gene real-time PCR assay for the detection and quantification of Mycoplasma genitalium

We developed and validated a real-time quantitative polymerase chain reaction (qPCR) assay to determine Mycoplasma genitalium bacterial load in endocervical swabs, based on amplification of the pdhD gene which encodes dihydrolipoamide dehydrogenase, using the Rotor-Gene platform. We first determined the qPCR assay sensitivity, limit of detection, reproducibility and specificity, and then determined the ability of the qPCR assay to quantify M. genitalium in stored endocervical specimens collected from Zimbabwean women participating in clinical research undertaken between 1999 and 2007. The qPCR assay had a detection limit of 300 genome copies/mL and demonstrated low intra- and inter-assay variability. The assay was specific for M. genitalium DNA and did not amplify the DNA from other mycoplasma and ureaplasma species. We quantified M. genitalium in 119 of 1600 endocervical swabs that tested positive for M. genitalium using the commercial Sacace M. genitalium real-time PCR, as well as 156 randomly selected swabs that were negative for M. genitalium by the same assay. The M. genitalium loads ranged between < 300 and 3,240,000 copies/mL. Overall, the qPCR assay demonstrated good range of detection, reproducibility and specificity and can be used for both qualitative and quantitative analyses of M. genitalium in endocervical specimens and potentially other genital specimens.     

Serologic and molecular evidence for Testudinid herpesvirus 2 infection in wild Agassiz's desert tortoises, Gopherus agassizii

Following field observations of wild Agassiz's desert tortoises (Gopherus agassizii) with oral lesions similar to those seen in captive tortoises with herpesvirus infection, we measured the prevalence of antibodies to Testudinid herpesvirus (TeHV) 3 in wild populations of desert tortoises in California. The survey revealed 30.9% antibody prevalence. In 2009 and 2010, two wild adult male desert tortoises, with gross lesions consistent with trauma and puncture wounds, respectively, were necropsied. Tortoise 1 was from the central Mojave Desert and tortoise 2 was from the northeastern Mojave Desert. We extracted DNA from the tongue of tortoise 1 and from the tongue and nasal mucosa of tortoise 2. Sequencing of polymerase chain reaction products of the herpesviral DNA-dependent DNA polymerase gene and the UL39 gene respectively showed 100% nucleotide identity with TeHV2, which was previously detected in an ill captive desert tortoise in California. Although several cases of herpesvirus infection have been described in captive desert tortoises, our findings represent the first conclusive molecular evidence of TeHV2 infection in wild desert tortoises. The serologic findings support cross-reactivity between TeHV2 and TeHV3. Further studies to determine the ecology, prevalence, and clinical significance of this virus in tortoise populations are needed.     

复合探针荧光定量PCR方法的建立

为了对特定基因进行实时检测,根据荧光能量转移(FRET)原理,设计及合成了一种新的FRET复合探针,该探针由一条长的荧光杂交探针和短的淬灭探针构成,其中荧光探针5′端接一荧光素分子,3′端接一延伸阻断分子磷酸,淬灭探针3′端连接一个淬灭分子对甲基红,淬灭探针与荧光探针5′端互补,无模板时,该探针杂交形成复合探针。无荧光产生,当有模板时,荧光探针与模板杂交,荧光不能被淬灭,产生的荧光与模板量成正比。根据复合探针的反应原理,研究了该探针的FRET性质及影响因素包括淬灭探针及扩增片段长度、荧光探针与淬灭探针的合适比例及镁离子浓度。实验结果显示淬灭探针及扩增片段长度对复合探针的作用有明显的影响,本实验采用淬灭探针长21个核苷酸,扩增片段长127bp,荧光探针与淬灭探针的合适比例为1：1,镁离子浓度为3mmo1／L,可获得最佳的反应体系;该复合探针合成简单,淬灭彻底,具有良好的准确性与特异性,敏感性达10^2拷贝,并具有较宽的动力学定量范围,可对10^2—10^9拷贝范围内的待检样品进行准确的定量。复合探针技术可应用于病毒感染水平、转基因拷贝数及单核苷酸多态性等检测。     

Nanoliter scale PCR with TaqMan detection.

We monitored PCR in volumes of the order of 10 nl in glass microcapillaries using a fluorescence energy transfer assay in which fluorescence increases if product is made due to template-dependent nucleolytic degradation of an internally quenched probe (TaqMan assay). This assay detected single starting template molecules in dilutions of genomic DNA. The results suggest that it may be feasible to determine the number of template molecules in a sample by counting the number of positive PCRs in a set of replicate reactions using terminally diluted sample. Since the assay system is closed and potentially automatable, it has promise for clinical applications.     

Comparison of various PCR methods for the detection of Chlamydia trachomatis, Ureaplasma urealyticum, and Mycoplasma hominis in women with impaired reproductive function

The comparative evaluation of the PCR test "Polimik" (Research and Production Firm "Litekh", Moscow) and the PCR test of the Novosibirsk Institute of Bioorganic Chemistry (NIBC) was carried out. The results obtained with the use of the PCR test "Polimik" and the PCR test of the NIBC of the detection of C. trachomatis and M. hominis coincided in 97.8% and 97.4% of cases. For U. urealyticum, the coincidence of the results of both PCR tests was 81.2%. Among women who visited gynecologists for reproductive function disturbances, the use of the PCR tests made it possible to detect C. trachomatis in 19 (5.5%) out of 343 cases, U. urealyticum in 96 (39.0%) out of 246 cases and M. hominis in 25 (16.9%) out of 148 cases. The results of the investigation revealed that the occurrence of C. trachomatis infection in Novosibirsk was comparable with that in other regions of the world among the low-risk groups of the population. The detection frequency of M. hominis and U. urealyticum with the use of the PCR tests showed that the occurrence of infections caused by these causative agents coincided with the data obtained in other countries.     

Detection of Mycoplasma hyopneumoniae by Air Sampling with a Nested PCR Assay

This article describes the first successful detection of airborne Mycoplasma hyopneumoniae under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 μm) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an M. hyopneumoniae-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 10⁴ times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne M. hyopneumoniae on 80% of farms where acute respiratory disease was present. No airborne M. hyopneumoniae was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and at a lower air humidity.     

鸡毒支原体强毒株与F疫苗株双重PCR检测方法的建立

目的建立能同时检测MG强毒株和F疫苗株的双重PCR技术。方法根据鸡毒支原体(MG)R株的PvpA基因序列和F疫苗株假定的α磷酸海藻糖酶基因序列,设计2对引物R1、R2和F1、F2,在单一PCR的基础上,建立检测MG强毒株和F疫苗株的双重PCR方法,并运用该双重PCR方法对临床样品进行检测。结果在330 bp和444 bp处分别出现预期的特异性DNA扩增条带,敏感性试验显示该体系能检测出0.45 ng的MG R株DNA和0.25 ng的MG F疫苗株DNA。临床样品MG强毒株阳性检出率为79.69%,高于常规分离培养鉴定法。结论成功建立检测两种毒株的双重PCR技术,为根除鸡群中MG野毒株、建立无MG的阴性鸡群提供新的技术手段。     

Development and clinical validation of a real-time PCR using a uni-molecular Scorpion-based probe for the detection of Mycoplasma pneumoniae in clinical isolates

Mycoplasma pneumoniae (Mp) is a frequent cause of Community Acquired Pneumoniae (CAP). The etiological role of Mp is usually suspected using serological assays, but the detection of specific anti-Mp antibodies becomes possible only 1-2 weeks after the primary infection. On the contrary, direct diagnosis using real-time PCR allows an efficient detection of Mp DNA in all the phases of the infection and particularly during early serum negative periods. In this study, we developed a novel Scorpion-probe real-time PCR-based assay. The probe's uni-molecular structure offers thermodynamic advantages owing to its kinetic reaction, providing faster performances compared to a TaqMan-based assay, but maintaining the same sensitivity and specificity. The Scorpion-based assay was employed on 388 clinical samples and compared with conventional qualitative PCR and serological tests. It was found more sensitive because it also allowed the detection of Mp in specimens found negative using classic qualitative PCR, but displaying seropositivity or a later seroconversion.