Automatized PCR evaluation of Mycobacterium tuberculosis complex in respiratory and nonrespiratory specimens

In this study, Mycobacterium tuberculosis complex isolates recovered from respiratory and nonrespiratory specimens with culture were evaluated using an automatized PCR method. Specimens with suspected tuberculous disease were decontaminated and concentrated using the standard N-acetyl-L-cysteine NaOH method and were inoculated onto glycerol-supplemented L?wenstein-Jensen media and BACTEC B12 vials. Forty-one specimens with typical colonies on solid media and 127 specimens identified as M. tuberculosis complex in a BACTEC system were selected as the study group. As the control group, 46 specimens without growth on either culture media were selected. The PCR results were positive in 33 (80.5%) and 87 (68.5%) samples that were culture-positive on solid and liquid media, respectively. All (100%) culture-negative specimens within the control group were also negative in the COBAS AMPLICOR Mycobacterium tuberculosis (MTB) PCR method. In conclusion, although it is a fast method for identifying M. tuberculosis complex isolates from clinical specimens, the COBAS AMPLICOR MTB PCR method is found to be less sensitive than culture techniques, we propose therefore that it should only be used in combination with culture results in the clinical diagnosis of tuberculosis.     

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

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

Multiplex PCR assay for simultaneous detection and differentiation of Mycobacterium tuberculosis, Mycobacterium avium complexes and other Mycobacterial species directly from clinical specimens

Aims: Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management. Methods and Results: Two hundred and thirty‐five samples from 145 clinically suspected cases of tuberculosis were processed for the detection of Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L‐J & BACTEC^TM MGIT‐960 culture and multiplex PCR tests. The multiplex PCR comprised of genus‐specific primers targeting hsp65 gene, Mycobacterium tuberculosis complex‐specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium avium complex‐specific primer pairs targeting 16S–23S Internal Transcribed Spacer sequences. The multiplex PCR developed had an analytical sensitivity of 10 fg (3–4 cells) of mycobacterial DNA. The multiplex PCR test showed the highest (77·24%) detection rate, while ZN smear examination had the lowest (20%) detection rate, which was bettered by L‐J culture (34·4%) and BACTEC^TM MGIT‐960 culture (50·34%) methods. The mean isolation time for M. tuberculosis was 19·03 days in L‐J culture and 8·7 days in BACTEC^TM MGIT‐960 culture. Using the multiplex PCR, we could establish M. tuberculosis + M. avium co‐infection in 1·3% HIV‐negative and 2·9% HIV‐positive patients. The multiplex PCR was also highly useful in diagnosing mycobacteraemia in 38·09% HIV‐positive and 15·38% HIV‐negative cases. Conclusions: The developed in‐house multiplex PCR could identify and differentiate the M. tuberculosis and M. avium complexes from other Mycobacterial species directly from clinical specimens. Significance and Impact of the Study: The triplex PCR developed by us could be used to detect and differentiate M. tuberculosis, M. avium and other mycobacteria in a single reaction tube.     

Evaluation of a low-cost calorimetric approach for rapid detection of tuberculosis and other mycobacteria in culture

Aims: The objective of this study was to evaluate the effectiveness of microcalorimetry in rapid detection of mycobacterium species using an inexpensive Isothermal microcalorimetry (IMC) instrument. In addition, we compared microcalorimetry with conventional monitoring techniques. Methods and Results: Isothermal microcalorimetry measures heat production rate and can provide rapid detection of living mycobacteria in clinical specimens. Using liquid medium showed that bacterial activity measured by IMC using a TAM Air^® agreed with the triphenyl tetrazolium chloride (TTC) assay. Using solid medium to enhance growth, fast‐growing mycobacteria detection was achieved between 26 and 53 h and slow‐growing mycobacteria detection was achieved between 54 and 298 h. In addition, the calorimetric data were analysed to estimate the growth rate and generation time of the mycobacteria monitored. Significance and Impact of the Study: Infections caused by mycobacteria are severe and difficult to treat. With 9·27 million new cases of tuberculosis in 2007, developing countries experience severe health and economic consequences owing to the lack of an affordable, fast detection method. Research‐grade IMC instruments are too expensive to use in developing countries. Our study demonstrates that less‐expensive instruments such as the TAM air ^® are adequate for mycobacteria detection and therefore establishes a clear proof of concept.     

Development of an IMS-PCR assay for the detection of Mycobacterium avium ssp. paratuberculosis in water

AIMS: To develop a sensitive detection method for Mycobacterium avium ssp. paratuberculosis (Map) in water by modifying and optimizing an existing immunomagnetic separation polymerase chain reaction (IMS-PCR) technique. METHODS AND RESULTS: Sterile distilled water (50 ml) spiked with 10(6) Map ml(-1) was subjected to either filtration (0.45 microm pore size) followed directly by IS900 PCR (method 1) or centrifugation (2500 g for 20 min) followed by IMS and IS900 PCR (method 2). Method 2 permitted the detection of Map, whereas method 1 did not. Method 2 was then optimized by adding different concentrations of Tween 80 (0.05, 0.1, 0.2, 0.4 and 0.6% v/v) to water samples spiked with Map (10(6)-1 CFU ml(-1)) prior to centrifugation, and assessing the impact of this action on the detection sensitivity of subsequent IMS-PCR. The optimum Tween 80 concentration was found to be 0.4%, which permitted the detection of 10 Map CFU ml(-1) in spiked water samples by IMS-PCR. CONCLUSIONS: This method will be used to determine the incidence of Map in water destined for domestic use in future studies. SIGNIFICANCE AND IMPACT OF THE STUDY: A sensitive method for the detection of Map in water involving addition of 0.4% Tween 80, centrifugation and IMS-PCR was developed.     

应用PCR技术检测结核分枝杆菌的临床分析

采用聚合酶链反应（Ｐｏｌｙｍｅｒａｎｓｅ Ｃｈｉａｎ Ｒｅａｔｉｏｎ,即ＰＣＲ）技术检测结核分枝杆菌Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｔｕｂｅｒｃｕｌｏｓｉｓ,一年多来共检测了１００例结核（肺、肾结核）患者的痰和尿液标本,结果ＰＣＲ检出阳性率为８１％,对照用储菌涂片抗酸染色法,阳性率为５８％,用常规培养法阳性率为２０％。而对５０例非结核患者的痰和尿液标本的检测,ＰＣＲ法仍有６％的阳性率,而用涂片或常规培     

Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory

Aims:  To facilitate efficient identification of commonly encountered mycobacteria species ( Mycobacterium tuberculosis , Mycobacterium avium , Mycobacterium intracellulare , Mycobacterium fortuitum complex , Mycobacterium chelonae/abscessus , Mycobacterium kansasii , Mycobacterium gordonae ) in high throughput laboratories, a 16s rDNA sequence based real-time PCR assay was developed and evaluated.
Methods and Results:  Oligonucleotide primers and hybridization probes were designed based on sequence differences of the mycobacterial 16S rDNA gene. This assay was evaluated with 1649 suspected non-tuberculosis mycobacterial isolates. Apart from 3 out of 40  M. avium isolates that showed false signal with M. intracellulare specific probe, 100% specificity was obtained for all tested probes. Assay sensitivity varied from 88·9 to 100% depending on species. Average cost for obtaining a definite identification was only USD 1·1 with an average turn around time of less than 3 days.
Conclusions:  A rapid, simple and inexpensive real-time PCR assay was developed for the identification of common encountered mycobacteria in a high throughput laboratory setting.
Significance and Impact of the Study:  With this assay, more than 80% of the clinically isolated nontuberculous mycobacteria could be identified in a highly cost effective manner. This helped to save resources for other laboratory activities especially in high throughput mycobacterial laboratories.     

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R² = 0.81, p < .001, biofouling R² = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.     

新型Taq Man-MGB探针在结核分枝杆菌实时PCR检测中的应用

为建立一种比现有方法敏感、准确性高、重复性好的结核分枝杆菌DNA定性定量检测方法 ,以TaqMan探针技术为基础 ,运用TaqMan MGB探针 ,实时检测临床标本中的结核分枝杆菌DNA .用来自临床标本的DNA及克隆于载体的IS6 1 1 0序列检测所建立方法的有效性 .结果显示 ,所建立方法的最低检测限度为 1个基因拷贝 反应 ,在每反应 1 0 0 ～ 1 0 8拷贝范围内 ,Ct 值同DNA量的对数呈线性关系 .同一模板不同时间或同一时间不同管内扩增 ,所得Ct 值恒定 .用该方法检测 37例结核分枝杆菌培养阳性的痰液标本 ,敏感度为 1 0 0 % ;用该方法检测 1 6例TB系列阴性参考品 ,特异性为1 0 0 % .结果表明 ,所建立的方法是用于结核分枝杆菌定性定量检测较理想的方法     

应用多重PCR方法检测石蜡包埋组织标本中的分枝杆菌DNA

应用多重PCR方法检测并鉴别石蜡包埋组织中的结核分枝杆菌复合体与非结核分枝杆菌DNA扩增片段类型 ,为结核分枝杆菌复合体感染与非结核分枝杆菌感染的病理学诊断提供一种补充的鉴别诊断方法。应用三对具有特异性的寡核苷酸引物 ,进行多重PCR扩增。这三对引物分别对应于分枝杆菌 6 5kD表面抗原、结核分枝杆菌插入序列IS6 1 1 0及人类β 珠蛋白基因的部分序列 ,其扩增产物分别为 3 83bp、1 2 3bp和 2 6 8bp。此种多重PCR方法检测的灵敏度为 0 6pg。经多重PCR扩增后进行凝胶电泳 ,结核分枝杆菌复合体 (结核分枝杆菌、牛型结核分枝杆菌、BCG)均可见 3 83bp、1 2 3bp片段 ,而非结核分枝杆菌 (鸟、龟、瘰疬、蟾蜍、堪萨斯、胞内、耻垢分枝杆菌 )仅见 3 83bp片段 (猿猴分枝杆菌与结核分枝杆菌复合体相同 )。与上述相比 ,分枝杆菌感染的临床标本分别增加了一条 2 6 8bp片段。对 2 0 9例临床初步诊断为淋巴结结核病人的石蜡包埋组织标本进行了多重PCR检测 ,1 93例病理诊断为淋巴结结核、结核性肉芽组织、结核性肉芽肿性炎症病人的标本 ,检测结果符合结核分枝杆菌复合体感…     

A Cas12a-based CRISPR interference system for multigene regulation in mycobacteria

Mycobacteria are responsible for a heavy global disease burden, but their relative genetic intractability has long frustrated research efforts. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but limitations of the prototypical Cas9-based platform, for example, in multigene regulation, remain. Here, we introduce an alternative CRISPRi platform for mycobacteria that is based on the minimal type V Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, reversible, can efficiently regulate essential genes and multiple genes simultaneously, and works as efficiently in infected macrophages as it does in vitro. Together, Cas12a-based CRISPRi provides a facile tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb), which will enable the development of synthetically lethal drug targets and the study of genes conditionally essential during infection.     

Simultaneous PCR amplification of two barley 5S rDNA sequence types in the same PCR reaction

Many researchers are currently using PCR technology to amplify individual members of multigene families, including 5S rDNA, for sequencing and related purposes. When members of the family differ in length, analyses would be facilitated if different units could be simultaneously and efficiently amplified. In the present paper we describe conditions that can be used to amplify simultaneously both the “long” and “short” 5S rDNA repeats found in barley (Hordeum vulgare L.).     

Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens

Aims: To develop a specific and highly sensitive loop-mediated isothermal amplification (LAMP) technique for the rapid detection of canine parvovirus (CPV) DNA directly in suspected faecal samples of dogs by employing a simple method of template preparation. Methods and Results: LAMP reaction was developed by designing two sets of outer and inner primers, which target a total of six distinct regions on VP2 gene of CPV. The template DNA was prepared by a simple boiling and chilling method. Of the 140 faecal samples screened by the developed LAMP and the conventional PCR assays, 104 samples (74·28%) were found positive by LAMP, whereas 81 samples (57·85%) were found positive by PCR. The specificity of the LAMP assay was tested by cross-examination of common pathogens of dogs and further confirmed by sequencing. The detection limit of the LAMP was 0·0001 TCID(50) ml(-1) , whereas the detection limit of the PCR was 1000 TCID(50) ml(-1) . Conclusions: The developed LAMP assay detects CPV DNA in faecal specimens directly within an hour by following a simple and rapid boiling and chilling method of template preparation. The result also shows that the developed LAMP assay is specific and highly sensitive in detecting CPV. Significance and Impact of the Study: The result indicates the potential usefulness of LAMP which is a simple, rapid, specific, highly sensitive and cost-effective field-based method for direct detection of CPV from the suspected faecal samples of dogs.     

Application of PCR for detection and identification of mycoplasma contamination in virus stocks

Summary A nested polymerase chain reaction (PCR) was used to detect and identify mycoplasma contaminants in viral stocks. The results of the PCR assay proved to be a sensitive and accurate indicator of the true status of the stock tested. Those samples positive by agar culture or Hoechst stain were also positive by PCR. Those samples that were inconclusive by Hoechst stain (10.05%) could be clearly determined to be mycoplasma positive or negative by PCR. The PCR assay also detected those fastidious species of mycoplasma that gave false negative results by the direct culture method. In many respects the PCR-based mycoplasma detection method described is superior to the agar culture and Hoechst staining detection methods. In this study, the PCR assay detected substantially more mycoplasma-positive viral stocks than did the agar culture assay. Due to its speed, sensitivity, and reliability, the PCR assay is of particular value in monitoring the process of removing mycoplasma from contaminated stocks. Furthermore, the PCR amplification products can be analyzed by restriction analysis to rapidly identify the species of the mycoplasma contaminating the stock tested.     

Detection of Mycobacterium avium ssp. paratuberculosis in cheese, milk powder and milk using IS900 and f57-based qPCR assays

Aims: To develop a quantitative PCR assay for sensitive and specific detection of Mycobacterium avium ssp. paratuberculosis (Map) in a range of dairy products. Methods and Results: TaqMan^® assays were designed to target the IS900 and f57 genetic elements of Map. Both real‐time PCR assays were integrated with the Adiapure^® Map DNA extraction kit and assessed separately for the detection/quantification of Map in spiked milk, Cheddar cheese and milk powder. Assays were validated against Cheddar cheese samples containing known concentrations of Map. The IS900 qPCR assay was significantly more sensitive than the assay based on the f57 primer/probe. At a threshold cycle value of 38, limits of detection (LOD) for the IS900 qPCR assay were 0·6 CFU ml^?1, 2·8 CFU g^?1 and 30 CFU g^?1 for artificially contaminated pasteurized milk, whole milk powder and Cheddar cheese, respectively. The respective LOD’s for the f57 assay were 6·2 CFU ml^?1, 26·7 CFU g^?1 and 316 CFU g^?1. Conclusion: The integrated Adiapure^® extraction – IS900 real time assay described is a sensitive, quantitative method for the detection of Map in dairy products. This is the first study to consider qPCR as a quantitative estimation of Map‐DNA in cheese and whole milk powder. Significance and Impact of the Study: The assay developed allows sensitive detection and quantification of Map DNA in a range of dairy products which is valuable for the screening and surveillance of this potential zoonotic organism.     

Development of a real-time PCR-based method for rapid differential identification of Mycobacterium species

AIMS: To develop a real-time PCR method for rapid differential identification of many clinically important mycobacteria to the species level. METHODS AND RESULTS: Eighteen Mycobacterium species that are considered clinically important were targeted for the identification. One primer pair and 21 pairs of hybridization probes (HybProbes) specific for the genus, species or complex were designed based on the rpoB gene sequences of mycobacteria. Twenty-five different Mycobacterium reference species were tested. In a single round of real-time PCR, all the nontuberculous mycobacteria (NTM) species tested were identified at the genus level and 16 of the 18 targeted species were differentially identified to the species or complex level during the amplification cycles; subsequent melting curve analysis allowed the specific identification of all the target species at the species or complex level without cross-reactivity with the other species. CONCLUSIONS: The developed real-time PCR assay rapidly identifies the NTM at the genus level and 18 clinically important Mycobacterium species at the species or complex level. SIGNIFICANCE AND IMPACT OF THE STUDY: This real-time PCR assay provides a useful tool for the rapid differentiation of most clinically important Mycobacterium species.     

快速可靠的双链PCR产物直接测序法

利用T7DNA聚合酶在低温下仍具较高活性的特点,在热变性后低温下进行测序反应,使用该方法对多种PCR产物进行序列分析均取得较好的结果．