Optimization of PCR in application of hot start <Emphasis Type="Italic">Taq</Emphasis> DNA polymerase for detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> with primers FER1-F and FER1-R1

There are two approaches in detection of bacterium Erwinia amylovora by PCR. One is based on detection of plasmid pEA29 and the other is based on detection of a chromosomal DNA sequence, specific for E. amylovora, in a sample. Since pathogenic strains without pEA29 have been isolated from the environment, methods based on this plasmid have been compromised and PCR methods based on chromosomal DNA species specific sequences became only reliable methods. PCR method with chromosomal primers FER1-F and FER1-R is currently the most reliable method due to its high sensitivity and specificity. The goal of this research is to make a significant improvement of the method by optimization of PCR in application of hot start DNA Taq polymerase, instead of wax, to obtain a hot start reaction. This enzyme, which is currently widely applied, can provide simpler achievement of hot start, saving labor and time and decreasing possibility of cross contamination of samples. Experiments showed that simple replacement of a regular recombinant Taq DNA polymerase by a hot start Taq DNA polymerase leads to complete failure of the reaction. Many optimization experiments had to be carried out to obtain an operational and reliable PCR which simultaneously has high sensitivity and specificity. Content of the reaction mixture, as well as temperature and time parameters of PCR, were significantly changed to achieve proper optimization.     

Detection of Laribacter hongkongensis using species-specific duplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (ISR)

Aims: For the rapid detection of Laribacter hongkongensis, which is associated with human community‐acquired gastroenteritis and traveller’s diarrhoea, we developed a duplex species‐specific PCR assay. Methods and Results: Full‐length of the 16S–23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR‐based sequencing. Two species‐specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species‐specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2·1 × 10^?2 ng μl^?1 genomic DNA which corresponds to 5000 CFU ml^?1. Conclusions: The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. Significance and Impact of the Study: This species‐specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.     

Detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> by novel chromosomal polymerase chain reaction primers

A new sensitive and specific method for the detection of Erwinia amylovora was developed. The method is based on the detection of a chromosomal DNA sequence specific for this bacterial species and enables detection of E. amylovora pathogenic strains, including recent isolates that lack plasmid pEA29 and thus cannot be detected by the previously popular PCR methods based on the detection of this plasmid. A species-specific random amplified polymorphic DNA (RAPD) marker was identified, cloned, and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. The E. amylovora specific sequence, 1269 bp long, was amplified in polymerase chain reaction and detected with electrophoresis in agarose gel stained with ethidium bromide. Amplification with other bacterial species did not produce any PCR product detectable by electrophoresis. Matching of the E. amylovora specific sequence to chromosomal DNA was confirmed by computer analysis of the E. amylovora genome. A consistent sensitivity limit of the method was 3 CFU/reaction, and in some cases it was possible to detect 0.6 CFU/reaction. Due to its high sensitivity and specificity, our method of E. amylovora detection is currently the most reliable, taking into account that the reliability of PCR methods based on plasmid pEA29 has been compromised by the isolation of pathogenic E. amylovora strains that lack this plasmid.     

Laboratory evaluation of the rapid diagnostic tests for the detection of Vibrio cholerae O1 using diarrheal samples

BackgroundCholera, an acute diarrheal disease is a major public health problem in many developing countries. Several rapid diagnostic tests (RDT) are available for the detection of cholera, but their efficacies are not compared in an endemic setting. In this study, we have compared the specificity and sensitivity of three RDT kits for the detection of Vibrio cholerae O1 and compared their efficiency with culture and polymerase chain reaction (PCR) methods.MethodsFive hundred six diarrheal stool samples collected from patients from two different hospitals in Kolkata, India were tested using SD Bioline Cholera, SMART-II Cholera O1 and Crystal-VC RDT kits. All the stool samples were screened for the presence of V. cholerae by direct and enrichment culture methods. Stool DNA-based PCR assay was made to target the cholera toxin (ctxAB) and O1 somatic antigen (rfb) encoding genes. Statistical evaluation of the RDTs has been made using STATA software with stool culture and PCR results as the gold standards. The Bayesian latent class model (LCM) was used to evaluate the diagnostic tests in the absence of the gold standard.ResultsInvolving culture technique as gold standard, the sensitivity and specificity of the cholera RDT kits in the direct testing of stools was highest with SAMRT-II (86.1%) and SD-Cholera (94.4%), respectively. The DNA based PCR assays gave very high sensitivity (98.4%) but the specificity was comparatively low (75.3%). After enrichment, the high sensitivity and specificity was detected with SAMRT-II (78.8%) and SD-Cholera (99.1%), respectively. Considering PCR as the gold standard, the sensitivity and specificity of the RDTs remained between 52.3–58.2% and 92.3–96.8%, respectively. In the LCM, the sensitivity of direct and enrichment testing was high in SAMRT-II (88% and 92%, respectively), but the specificity was high in SD cholera for both the methods (97% and 100%, respectively). The sensitivity/specificity of RDTs and direct culture have also been analyzed considering the age, gender and diarrheal disease severity of the patients.ConclusionOverall, the performance of the RDT kits remained almost similar in terms of specificity and sensitivity. Performance of PCR was superior to the antibody-based RDTs. The RTDs are very useful in identifying cholera cases during outbreak/epidemic situations and for making them as a point-of-care (POC) testing tool needs more improvement.     

Development of a loop-mediated isothermal amplification assay for detection of <Emphasis Type="Italic">Cronobacter</Emphasis> spp. (<Emphasis Type="Italic">Enterobacter sakazakii</Emphasis>)

Contamination of Cronobacter spp. (Enterobacter sakazakii) in infant formulas and other food products is a severe problem. Here a loop-mediated isothermal amplification (LAMP) assay was developed for rapidly detecting Cronobacter spp. in powdered infant formula. Sequences of 16S/23S rDNA internal intergenic spacer of Cronobacter spp. were used as the target template to design LAMP primers. The detection outcome can be evaluated by the white precipitate or the fluorescence intensity under ultraviolet irradiation, both visible to naked eyes. The sensitivity and specificity of the LAMP assay was further analyzed in comparison with that of regular PCR and real time quantitative PCR. The results showed that all of Cronobacter spp. strains display positive reaction to the detections while all of the non-Cronobacter spp. strains were negative, and that the LAMP assay exhibits a high sensitivity of 9.1 fg/μL (The sensitivity of regular PCR and real time quantitative PCR is 91 and 9.1 pg/μL, respectively.). The amplified reaction could be accomplished in about 1 h, with the results visible to naked eyes. Hence, the LAMP assay developed by this study can provide a rapid and simple approach for the detection of Cronobacter spp. in infant formula.     

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

Simple and rapid detection of Salmonella serovar Enteritidis under field conditions by loop‐mediated isothermal amplification

Aim: The objective of this study is to develop a serovar‐specific loop‐mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions. Methods: A set of six specific primers was designed with Salmonella Enteritidis DNA as the target. LAMP conditions were optimized by incubating the target DNA with the Bst DNA polymerase large fragment in a simple water bath. The sensitivity and specificity of LAMP was then compared with those of fluorescent quantitative real‐time polymerase chain reaction (FQ‐PCR). Results: The results were as follows. (1) Serovar‐specific Salmonella Enteritidis DNA was amplified at 65°C in as early as 20 min in a water bath. (2) A colour change visible to the naked eye indicated a positive amplification reaction. (3) The detection limit of the LAMP assay was 4 copies μl^?1; thus, the sensitivity and specificity of this assay is similar to those of the FQ‐PCR. Conclusions: LAMP is a high‐throughput detection technique with high sensitivity, specificity, and simplicity; these factors make it suitable for specifically detecting Salmonella Enteritidis under field conditions and in laboratory settings. Thus, LAMP eliminates the need for complicated equipment and technical training in the detection of this specific serovar. Significance and impact of the study: This is the first study involving the use of LAMP to detect Salmonella serovar‐specific DNA sequences. It is also the first to report an ideal method of distinguishing between Salmonella Enteritidis and other Salmonella under field conditions.     

Molecular Typing of Cyst‐Forming Nematodes Globodera pallida and G. rostochiensis,Using Real‐Time PCR and Evaluation of Five Methods for Template Preparation

Globodera pallida and G. rostochiensis are two cyst‐forming nematodes known to infest potato crops, causing severe economic losses worldwide. In this study, a real‐time TaqMan PCR assay was developed and optimized for the simultaneous detection of G. pallida and G. rostochiensis. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Four different DNA extraction methods and one rapid crude template‐preparation procedure were compared in terms of extraction purity, efficiency for PCR applications, utility and cost. Extraction methods A and B included two commercially available kits that utilize silica columns and magnetic beads, respectively. Method C was based on DNA isolation using Chelex resin, and method D was a standard chemistry in‐house protocol. Procedure E included the direct use of crude mixture composed of disrupted cysts in Tris–EDTA buffer. The multiplex TaqMan PCR assay successfully discriminated the two nematode species from all reference cyst samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional (Co) PCR tests, the overall Dsp and Dse were lower and estimated at 94 and 87% for G. pallida, and 97 and 88% for G. rostochiensis, respectively. Spectrophotometric results showed that DNA extraction methods A, B and C yielded the purest DNA and gave the lowest mean C_t values as well as the most consistent results in Co PCR. Alternative crude preparation method E resulted in statistically similar and C_t values consistent with those obtained with methods A to C when tested by TaqMan PCR. The developed assay, using crude template‐preparation E, allows the simple, accurate and cost‐effective testing of a large number of cyst samples and can be applied in surveys and certification schemes.     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.     

Developing noninvasive methodologies to assess koala population health through detecting Chlamydia from scats

Wildlife diseases are a recognized driver of global biodiversity loss, have substantial economic impacts, and are increasingly becoming a threat to human health. Disease surveillance is critical but remains difficult in the wild due to the substantial costs and potential biases associated with most disease detection methods. Noninvasive scat surveys have been proposed as a health monitoring methodology to overcome some of these limitations. Here, we use the known threat of Chlamydia disease to the iconic, yet vulnerable, koala Phascolarctos cinereus to compare three methods for Chlamydia detection in scats: multiplex quantitative PCR, next generation sequencing, and a detection dog specifically trained on scats from Chlamydia‐infected koalas. All three methods demonstrated 100% specificity, while sensitivity was variable. Of particular interest is the variable sensitivity of these diagnostic tests to detect sick individuals (i.e., not only infection as confirmed by Chlamydia‐positive swabs, but with observable clinical signs of the disease); for koalas with urogenital tract disease signs, sensitivity was 78% with quantitative PCR, 50% with next generation genotyping and 100% with the detection dog method. This may be due to molecular methods having to rely on high‐quality DNA whereas the dog most likely detects volatile organic compounds. The most appropriate diagnostic test will vary with disease prevalence and the specific aims of disease surveillance. Acknowledging that detection dogs might not be easily accessible to all, the future development of affordable and portable “artificial noses” to detect diseases from scats in the field might enable cost‐effective, rapid and large‐scale disease surveillance.     

A multiple PCR‐LDR assay for identification of two invasive cryptic species of whiteflies Bemisia tabaci

The MEAM1 and MED species of the cryptic species complex Bemisia tabaci are important invasive pests that cause tremendous crop losses worldwide. A rapid and highly reliable molecular technique is necessary to identify these species because they are morphologically indistinguishable. Therefore, a multiple polymerase chain reaction coupled with a ligase detection reaction (PCR‐LDR) that was based on polymorphisms in the mitochondrial cytochrome oxidase I (mtCOI) gene of B. tabaci was developed to distinguish the two cryptic species. An assessment of the method indicated that PCR‐LDR provided high specificity and sensitivity in discriminating MEAM1 (SHB) and MED (SHQ) whiteflies. In field tests, PCR‐LDR genotyping was performed in one 96‐well plate to identify 93 individuals collected from 8 districts in the suburbs of Shanghai. Complete concordance was observed between PCR‐LDR and sequencing methods. The method was used to confirm that MEAM1 and MED were found in two districts, but only the MED was found in the other six districts. PCR‐LDR, which is a transplantable platform, provides an alternative method for species identification of B. tabaci at low cost.     

Sensitive and rapid detection of Karenia mikimotoi (Dinophyceae) by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was developed to detect the genomic DNA of Karenia mikimotoi using a set of four specific primers based on a ribosomal DNA internal transcribed spacer (ITS). The sensitivity of this LAMP assay was 100-fold higher than regular PCR, and its specificity was validated using other algae as a comparison. Two visual detection approaches were feasible to interpret the positive or negative results. This technology may have the potential to aid in forecasting red-tides on the scene because of its high sensitivity, specificity and rapid detection.     

A real‐time PCR assay for improved rapid,specific detection of Cryphonectria parasitica

Cryphonectria parasitica, an ascomycete fungus, is the causal agent of chestnut blight. This highly destructive disease of chestnut trees causes significant losses, and is therefore a regulated pathogen in Europe. Existing methods for the detection of C. parasitica include morphological identification following culturing, or PCR; however, these are time‐consuming resulting in delays to diagnosis. To allow improved detection, a new specific real‐time PCR assay was designed to detect C. parasitica directly from plant material and fungal cultures, and was validated according to the European Plant Protection Organisation (EPPO) standard PM 7/98. The analytical specificity of the assay was tested extensively using a panel of species taxonomically closely related to Cryphonectria, fungal species associated with the hosts and healthy plant material. The assay was found to be specific to C. parasitica, whilst the analytical sensitivity of the assay was established as 2 pg µL^?1 of DNA. Comparative testing of 63 samples of naturally infected plant material by the newly developed assay and traditional morphological diagnosis demonstrated an increased diagnostic sensitivity when using the real‐time PCR assay. Furthermore the assay is able to detect both virulent and hypovirulent strains of C. parasitica. Therefore the new real‐time PCR assay can be used to provide reliable, rapid, specific detection of C. parasitica to prevent the accidental movement of the disease and to monitor its spread.     

Loop‐mediated isothermal amplification for the rapid detection of Gardnerella vaginalis

The aim of this study was to develop a method for the rapid detection of Gardnerella vaginalis, which is proposed to play a key role in the pathogenesis of bacterial vaginosis. Specific loop‐mediated isothermal amplification (LAMP) primers were designed and used to detect target DNA within 45 min under isothermal conditions. Comparative screening indicated that the LAMP assay is superior to PCR in terms of rapidity, and is equivalent in sensitivity and specificity. This LAMP assay can be used for rapid screening and detection of G. vaginalis in vaginal samples; the limit of detection is 10 fg DNA.

     

A Species-Specific Polymerase Chain Reaction Assay for Rapid and Sensitive Detection of <Emphasis Type="Italic">Colletotrichum</Emphasis><Emphasis Type="Italic">capsici</Emphasis>

Colletotrichum capsici is an important fungal species that causes anthracnose in many genera of plants causing severe economic losses worldwide. A primer set was designed based on the sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a conventional PCR assay. The primer set (CcapF/CcapR) amplified a single product of 394 bp with DNA extracted from 20 Mexican isolates of C. capsici. The specificity of primers was confirmed by the absence of amplified product with DNA of four other Colletotrichum species and eleven different fungal genera. This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate and cultivate the fungus in order to identify it. The sensitivity of detection with this PCR method was 10 pg of genomic DNA from the pathogen. This is the first report of a C. capsici-specific primer set. It allows rapid pathogen detection and provides growers with a powerful tool for a rational selection of fungicides to control anthracnose in different crops and in the post-harvest stage.     

Comparison of <Emphasis Type="Italic">mecA</Emphasis> Polymerase Chain Reaction With Phenotypic Methods for the Detection of Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In the present study, several conventional methods to detect methicillin-resistant Staphylococcus aureus (MRSA) were compared with polymerase chain reaction (PCR) detection of mecA gene–positive isolates. Cefoxitin E-test was also evaluated as a possible phenotypic method of MRSA detection. Oxacillin agar screen and PBP2′ latex agglutination methods were found to be more sensitive than oxacillin and cefoxitin disk-diffusion methods. Cefoxitin disk diffusion was found to be the most specific. A combination of oxacillin agar screening with cefoxitin disk diffusion, or oxacillin disk diffusion with PBP2′, improved sensitivity and specificity. Cefoxitin E-test with the current break points had low sensitivity and specificity (33.3% and 75%, respectively) for the detection of MRSA. However, changing the break points to ≤ 4 μg/ml and to ≥ 6 μg/ml for sensitive and resistant, respectively, greatly improved both. Changing the 30-μg cefoxitin disk-diffusion break points to ≤ 21 mm for resistant slightly improved sensitivity but had no effect on specificity. It was therefore concluded that the use of more than one screening method is necessary to detect all MRSA isolates in clinical settings.     

New bioluminescence pyrophosphate assay for high‐sensitivity detection of food‐borne pathogens

Traditional methods of identifying food‐borne pathogens are time consuming and laborious, so innovative methods for their rapid identification must be developed. Testing for bioluminescence pyrophosphate is a convenient and fast method of detecting pathogens without complex equipment. However, the sensitivity of the method is not as high as that of other methods, and it has a very high detection limit. In this study, the method was optimized to improve its sensitivity. The shortcomings of the method were first identified and corrected using dATPαS instead of dATP for the polymerase chain reaction (PCR), therefore reducing the background signal. Also, when the DNA template extracted from the food‐borne pathogens was purified, the new bioluminescence pyrophosphate assay had a limit of detection of <10 copy/μl or 10 colony‐forming units/ml, and its sensitivity was higher than that of fluorescent real‐time quantitative PCR. Moreover, a single copy of a food‐borne pathogen could be detected when a single DNA template was included in the PCR. Salmonella was detected in and isolated from 60 samples of broiler chicken, and the accuracy of the results was verified using a culture method (GB 4789.4–2010). These results showed that the new bioluminescence pyrophosphate assay has the advantages of an intuitive detection process, convenient operation, and rapid measurements. Therefore, it can be used for the rapid detection of pathogenic bacteria and probiotics in various fields.     

Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real‐time PCR assay

Aim: The aim of this study was to develop a multiplex real‐time PCR assay for the identification and discrimination of Erysipelothrix rhusiopathiae, tonsillarum and Erysipelothrix sp. strain 2 for direct detection of Erysipelothrix spp. from animal specimens. Methods and Results: A primer set and three species‐specific probes with different end labelling were designed from the noncoding region downstream of the 5S rRNA coding region. The sensitivity, specificity and repeatability of the assay were validated by analysing 27 Erysipelothrix spp. reference serotype strains and ten septicemia‐associated non‐Erysipelothrix spp. bacterial isolates. Cross‐reactivity with Erysipelothrix sp. strain 1 was not observed with any of the primer probe combinations. The detection limit was determined to be <10 colony forming units and as low as one genome equivalent per PCR . Further evaluation of the Erysipelothrix spp. multiplex PCR was performed by comparing an enrichment isolation culture method and a conventional differential PCR on 15 samples from pigs experimentally inoculated with Erysipelothrix spp. and 22 samples from pigs with suspected natural infection. Conclusion: The multiplex real‐time PCR assay was found to be simple, rapid, reliable, specific and highly sensitive. Significance and Impact of the Study: The developed real‐time multiplex PCR assay does not require cumbersome and lengthy cultivation steps prior to DNA extraction, obtained comparable results to enrichment isolation, and will be useful in diagnostic laboratories for rapid detection of Erysipelothrix spp.