Polymerase Spiral Reaction Assay Research Articles

Development, evaluation and application of a polymerase spiral reaction (PSR) based assay on accurate detection of Vibrio parahaemolyticus viable cells and virulence factors from rice product

Vibrio parahaemolyticus is a predominate cause of microbiological food safety. This study aimed to develop a polymerase spiral reaction (PSR) based assay for rapid and visual detection on V. parahaemolyticus. Firstly, the specificity and conservativity of species-specific target tlh gene were determined. Secondly, targeting tlh and virulence factors tdh and trh, PSR assay was developed and optimized using constructed positive plasmid controls and standard strain. Visualized result determination using fluorescent dye was applied to avoid false positive issues. Thirdly, 44 strains were used for specificity evaluation. Fourthly, pure and mixed species artificial contamination systems were included to test its applicability. In addition, a propidium monoazide (PMA)-PSR assay was set up to determine viable but non-culturable (VBNC) cells. Target tlh gene acquired high specificity and PSR assay was optimized to carry out at 65ᵒC for 1 h. Detection limit of PSR assay in purified genomic DNA, plasmid and artificially contaminated food samples were 5.31–53.1 pg/μL, 100 copies, and 103 CFU/mL, respectively, demonstrating the sensitivity as 100-fold higher than regular PCR. With PMA-PSR assay, VBNC cells were accurately determined without the disruption of dead cells and food compositions. Thus, PSR is applicable in detection of V. parahaemolyticus viable cells and virulence factors.

Rapid and visual detection of Listeria monocytogenes based on polymerase spiral reaction in fresh-cut fruit

Widespread foodborne pathogen Listeria monocytogenes exhibits a pronounced fatality rate among individuals with compromised immune systems, the elderly, and pregnant women. A simple and user-friendly approach is required to identify L. monocytogenes. A distinctive thermostatic nucleic acid amplification technology called polymerase spiral reaction (PSR) has been extensively employed in the identification of foodborne pathogens. In the study, a straightforward and intuitive approach for identifying L. monocytogenes in fresh-cut fruit was established using PSR in conjunction with hydroxy naphthol blue (HNB). Following optimization, the system's primary constitutes-betaine, dNTP, and MgSO4 were found to be 0.5 M, 1.0 mM, and 6 mM, respectively. Six strains of non- L. monocytogenes were used to test the assay's specificity. The PSR assay performed at 65 °C for 50 min demonstrated the capability to detect L. monocytogenes at level as low as 1 × 10−4 ng/μL DNA per tube and 5.1 × 101 CFU/g of freshly cut fruit. Notably, the sensitivity of this approach surpassed that of PCR by10-fold (1 × 10−3 ng/μL). In order to detect L. monocytogenes, the PSR assay was designed to be rapid, accurate, effective, and apparatus free. This innovative assay has provided researchers a fresh perspective on identification harmful microorganisms.

Visual detection of Staphylococcus aureus based on immunomagnetic separation and polymerase spiral reaction

Staphylococcus aureus (S. aureus) is widely distributed in the environment, which can easily lead to food contamination. Thus, it is essential to develop rapid and on-site detection of S. aureus. In the present study, an intuitive colorimetric method based on phage display technology (PDT), immunomagnetic separation (IMS), and polymerase spiral reaction (PSR) was successfully established for S. aureus detection. Peptide-2 (P2, MNVTFTT) with high affinity and specificity for S. aureus was screened by PDT. The formed P2-magnetic beads (MBs) were able to effectively enrich and isolate S. aureus from complex matrices under applied magnetic field. Then a PSR assay was developed for signal amplification and visualization and performed in a thermostatic metal bath using specific primers targeting nuc gene. The results could be observed by naked eyes with a significant color change from purple to blue. The newly developed IMS-PSR assay enabled detection of S. aureus at levels as low as 84 CFU/mL (1.5 pg) in spiked milk sample within 3 h. With all these benefits, it shows promise for rapid on-site visual detection of S. aureus.

Development of novel isothermal-based DNA amplification assay for detection of pig tissues in adulterated meat.

For the first time, we describe an innovative polymerase spiral reaction (PSR) assay for the rapid, simple, and accurate detection of pig tissues or pork in adulterated meat including heat-treated and processed ones. The PSR assay specifically targeting the mitochondrial cytochrome b (cyt-b) gene of the pig was successfully optimized permitting assay results in 65min time. The developed detection method was 100% specific amplifying only the cyt-b gene and displaying negative results with all the tested non-pork meats. The sensitivity of the developed PSR (760fg porcine DNA) was tenfold better than the end-point PCR and able to detect heat-treated (121°C) and adulterated (0.5% pork in beef) meat and processed pork products such as sausages, salami, meatball, soup, curry, etc. The developed PSR-based method can be used for point-of-care detection with minimum instrumentation and technical expertise to guarantee instant clearance of exported and imported meat products. This is the first time that PSR has been adapted for food authenticity purposes.

Development and application of multiple polymerase spiral reaction (PSR) assays for rapid detection of methicillin resistant Staphylococcus aureus and toxins from rice and flour products

Methicillin-Resistant Staphylococcus aureus (MRSA) is an important foodborne pathogen and produces a variety of toxins causing serious illnesses. This study aimed at developing a rapid detection assay for MRSA and toxins targeting on two housekeeping genes (mecA and femA) and 5 virulence factors (sea, seb, sec, see and pvl) using the polymerase spiral reaction (PSR), compared with PCR. For naked-eye result inspection, calcein was added and the PSR reaction can be completed at 65ᵒC within 60 min. The PSR assay was subjected to optimization, evaluation, and application in 3 different types of food sample made from rice and flour, designated rice and flour product. The limit of detection of PSR assay for the mecA, femA, sea, seb, sec, see and pvl genes was 4.2 pg/μL, 0.42 pg/μL, 630 pg/μL, 63 pg/μL, 12.3 pg/μL, 1.015 pg/μL and 53 pg/μL, respectively, which was higher than the sensitivity of PCR. In rice and flour products, the detection limit of PSR assay was 104 CFU/mL for mecA, and 103 CFU/mL for femA, sea, seb, sec, see and pvl genes. In conclusion, optimized PSR assay serves as an efficient tool for rapid, cost-effective and accurate testing of MRSA and is suitable for point-of-care detection.

A Novel and Rapid Isothermal Nucleic Acid Based Detection Assay of Vibrio cholerae by Polymerase Spiral Reaction (PSR) in Emergency Situations.

The purpose of this survey was to develop a novel and rapid isothermal nucleic acid based detection assay of Vibrio cholerae by polymerase spiral reaction (PSR) in emergency situations. The current study was conducted in Baqiyatallah University of Medical Sciences, Tehran, Iran in 2021. The conserved ctxA gene sequence of V. cholerae was used as a target of designed two pairs of primers. Amplification of nucleic acids performed under isothermal temperature of 65 °C in 55 min by using Bst DNA polymerase. PSR amplified products were real-time visualized under UV transilluminator and also on agarose gel electrophoresis. Seven non- V. cholerae bacteria were negative for detection, which indicated the specificity of PSR assay was 100%. A 10- fold serial dilution of V. cholerae genomic DNA was subjected to conventional polymerase chain reaction (PCR) and real-time PCR to compare their sensitivities with PSR. The detection limit of PSR was 3 × 10-5 ng/μL within 60 min, which 100-fold higher than that of PCR (3 × 10-3 ng/μL), but the sensitivity of real-time PCR was found as same as it. The PSR assay developed in this study can provide a simple, cost-effective, rapid, and precise diagnosis technique in endemic cholera outbreaks, especially in low-income with limited access provinces.

Polymerase Spiral Reaction (PSR) as a novel rapid colorimetric isothermal point of care assay for detection of Trypanosoma evansi genomic DNA

Polymerase spiral reaction (PSR) opens new avenues for specific diagnosis of pathogens known for cryptic infection at field level and its application is still unexplored in the field of parasitology. The present study aimed to explore and optimize colorimetric based PSR technique for the detection of Trypanosoma evansi in the blood of the host by targeting the 196bp Invariable Surface Glycoprotein (ISG) gene of Trypanosoma evansi. The specificity of the test was determined against Theileria equi, Theileria annulata, Babesia caballi, Burkholderia mallei and Equine herpes virus. The T. evansi DNA was extracted from purified parasites and serially diluted from 2.8ng to 2.8 × 10−8 pg. The detection limit of PSR was found to be as low as 2.8 × 10-6 pg of T. evansi DNA, which will aid in detection of Surra infection. The duration of reaction for determination of result of field sample is 1h and result can be read by naked eyes. In addition, PSR assay was also performed on DNA extracted from 28 field equine samples; out of which 1 was found positive by microscopy and ISG-196 targeted PCR assay and 2 were recorded positive by PSR assay. Data generated shows colorimetric PSR is convenient, rapid, sensitive and specific tool for diagnosis and monitoring of Surra infection in livestock at field level. Further, visual PSR assay has wide scope for application in government policies aimed at detection of early infection, sub-clinical cases, drug-efficacy studies, control and elimination of Surra organism from livestock animals.

Development of BruAb2_0168 based isothermal polymerase spiral reaction assay for specific detection of Brucella abortus in clinical samples

Bovine brucellosis, predominantly caused by Brucella abortus is one of the most neglected zoonotic diseases causing severe economic losses in the dairy industry. The early and precise diagnosis of the disease is required to reduce the transmission of infection in humans as well as animals. In the current study, a rapid and novel isothermal amplification-based polymerase spiral reaction (PSR) was developed for the specific detection of Brucella abortus by targeting the BruAb2_0168 gene. The assay could be conducted at 65 °C in a water bath and results can be obtained after 60 min. The detection limit of the PSR assay was found to be 1.33 fg. The sensitivity of the assay was found to be 104 fold higher than conventional PCR and equivalent to real-time PCR (RT-PCR). The assay didn't exhibit cross-reaction with selected pathogenic non-Brucella bacteria and Brucella spp. other than B. abortus. Forty clinical samples were also tested using this novel assay and it was able to detect 25 samples as positive, however, conventional PCR could detect the targeted organism in 22 samples only.To the extent of our knowledge, this is the first report towards the development of a PSR assay for specific detection of B. abortus. The assay can be used as a quick, sensitive and accurate test for the diagnosis of bovine brucellosis in the field setting. Relatively one of the paradigm-shifting aspects of this assay would be it does not require any expensive equipment and the results can be easily visualized by the unaided eye, therefore making PSR a valuable diagnostic tool in field conditions.

Development of a novel visual detection technique for Campylobacter jejuni in chicken meat and caecum using polymerase spiral reaction (PSR) with pre-added dye

Rapid detection and efficient removal of Campylobacter jejuni contaminated chicken-based food products from the food chain have become decisive for preventing outbreaks. Here, we report a novel, rapid, simple and sensitive polymerase spiral reaction (PSR) assay for screening chicken samples for contamination of C. jejuni. Our method specifically amplified the hipO gene of C. jejuni and involved cost-effective three-step centrifugation method for DNA extraction and pre-addition of HNB dye for effortless visualization. PSR, real-time PCR and conventional PCR were able to detect 50 fg, 500 fg and 5 pg C. jejuni DNA per tube, respectively. In chicken meat and caecum, the detection limits of PSR without any enrichment, after including 12 h enrichment and 24 h enrichment steps were 16 × 102 CFU/g, 160 CFU/g and 16 CFU/g, respectively which could be attained in 90 min, 13.5 h and 25.5 h, respectively. The field applicability of the PSR assay was evaluated through a field sampling study by testing 75 chicken meat and caecum samples. This assay permits timely reporting of results without the need of trained personnel and sophisticated equipments as it only relies on simple enrichment, water bath amplification and visual interpretation through colour change. This PSR assay can be a handy tool to screen C. jejuni contamination in chicken products and might subsequently help in reducing the incidence of foodborne C. jejuni infections. To our knowledge, this is the primary report to employ PSR for the detection of C. jejuni.

Rapid Detection of Hepatitis B Virus in Blood Samples Using a Combination of Polymerase Spiral Reaction With Nanoparticles Lateral-Flow Biosensor.

A rapid, highly sensitive, and robust diagnostic technique for point-of-care (PoC) testing can be developed using the combination of the nanoparticle-based lateral flow biosensors (LFB) and isothermal nucleic acid amplification technology. Here, we developed a polymerase spiral reaction (PSR) containing FITC-labeled DNA probes coupled with the nanoparticle-based LFB assay (PSR-LFB) to detect the amplified products to detect HBV visually. Under the optimized conditions, the PSR assay involved incubation of the reaction mixture for 20 min at 63°C, followed by visual detection of positive amplicons using LFB, which would generate a red test line based on the biotin/streptavidin interaction and immunoreactions, within 5 min. A cross-reactivity test revealed that the developed PSR-LFB assay showed good specificity for HBV and could distinguish HBV from other pathogenic microorganisms. For the analytical sensitivity, the limit of detection (LoD) of PSR-LFB assay was recorded as 5.4 copies/mL of HBV genomic DNA, which was ten-times more sensitive than qPCR and loop-mediated isothermal amplification (LAMP). Additionally, all the HBV-positive (29/82) samples, identified using ELISA, were also successfully detected by the PSR-LFB assay. We found that the true positive rate of the PSR-LFB assay was higher than that of qPCR (100 vs. 89.66%, respectively), as well as the LAMP assay (100 vs. 96.55%, respectively). Furthermore, the integrated procedure could be completed in 60 min, including the processing of the blood samples (30 min), an isothermal reaction (20 min), and result visualization (5 min). Thus, this PSR-LFB assay could be a potentially useful technique for PoC diagnosis of HBV in resource-limited countries.

Development of a novel polymerase spiral reaction (PSR) assay for rapid and visual detection of Clostridium perfringens in meat

C. perfringens is a widespread foodborne pathogen and one of the major concerns in the meat industry. There is a need for a simple, rapid and equipment free detection system for C. perfringens as conventional anaerobic culture method is labour and resource intensive. Here, we applied a novel polymerase spiral reaction phenomenon to develop and evaluate an assay for effortless and visual detection of C. perfringens in meat foods employing pork as a representative model. Specificity of the assay was determined using 51 C perfringens and 20 non- C. perfringens strains. Analytical sensitivity of the developed test was 80 fg DNA per tube indicating 100 times more sensitivity than end-point PCR assay. The detection limits were 980 CFU/g and 9.8 × 104 CFU/g of pork for PSR and PCR assays, respectively. The operation time of the PSR assay including DNA extraction was 120 min. The developed PSR assay was accurate and effective in comparison to culture method, in detecting C. perfringens in 38 of 74 pork samples. Therefore the specificity, sensitivity, negative predictive value, positive predictive value and accuracy rate of the developed PSR assay were 100%. The developed PSR assay is easy to perform, rapid, affordable, permitting sophisticated-equipment free amplification and naked eye interpretation. This is the initial report in which the PSR assay was optimized for the detection of C. perfringens.

Development of a novel polymerase spiral reaction (PSR) assay for rapid and visual detection of Staphylococcus aureus in meat

Staphylococcus aureus is one of the most common and leading causes of food poisoning and also responsible for multifarious diseases in humans. Rapid and on-site detection of S. aureus is of specific importance in developing countries. In the present study, a polymerase spiral reaction (PSR) assay was developed for sensitive, rapid and visual detection of S. aureus and validated in meat using pork as a model. The specificity of the PSR assay was ascertained by using 18 S. aureus and 17 non-S.aureus strains. The PSR assay was 100- and 10-fold more sensitive than conventional end-point PCR and real-time PCR, respectively. The limit of detection of the PSR assay was 19.9 × 103 CFU/g of pork without enrichment and 19.9 CFU/g after 6 h enrichment. The detection of 19.9 CFU/g of pork was attained within 8 h. Real-world or field applicability of the developed assay was evaluated by screening 76 raw and processed pork samples. Seeing its rapidity, simplicity, cost-effectiveness, user-friendliness and sophisticated-equipment free nature, this assay has the prospective to turn into the assay of choice for regular detection of S. aureus in foods in less-advanced laboratories. To our knowledge, this is the first report to use PSR for the detection of S. aureus.

Development of a novel and rapid polymerase spiral reaction (PSR) assay to detect Salmonella in pork and pork products

The polymerase spiral reaction (PSR), a novel isothermal method for targeted DNA amplification, was effectively applied to detect Salmonella in artificially spiked pork. The specificity of the developed PSR was tested using 16 Salmonella and 15 non-Salmonella strains. The PSR assay was 10-fold more sensitive than conventional end-point PCR, having a sensitivity comparable to real-time PCR. The limit of detection of the developed assay was 4 × 103 per gram of pork without enrichment and 4 CFU per gram after a 6 h enrichment. The detection of 4 CFU per gram of pork was achieved within 8 h. The PSR assay was successful, and accurate in comparison to microbiological methods, in detecting Salmonella in 11 of 76 commercial pork samples. Therefore the positive predictive value, negative predictive value and accuracy rate of the developed assay were 100%. Considering its rapidity, user-friendliness, simplicity, cost-effectiveness and equipment-free nature, this PSR assay is a promising tool for the food industry for the detection of Salmonella and prevention of Salmonella outbreaks and recalls.

Rapid and visible detection of Mycoplasma synoviae using a novel polymerase spiral reaction assay

In this study, a rapid, specific, and sensitive detection assay for Mycoplasma synoviae (MS) was established using a polymerase spiral reaction (PSR) method. A pair of primers were designed according to the conserved region of the vlhA gene of MS, and PSR results were assessed using agarose gel electrophoresis and color rendering with a dye indicator. The optimum reaction temperature and time for PSR using the specific primers were 62°C and 40min in a water bath, respectively. The sensitivity of the PSR assay for MS detection was 100 times more than that of the polymerase chain reaction assay based on agarose gel electrophoresis results and color change detected by the naked eye. Further experiments demonstrated that the primers specifically detected MS and showed no cross-reaction with other prevalent avian pathogens. Clinical sample testing confirmed that the MS-PSR assay is simple, rapid, specific, and sensitive, and thereby very suitable for application and promotion in the field and laboratories of grassroots units.

Novel polymerase spiral reaction assay for the visible molecular detection of porcine circovirus type 3

BackgroundPorcine circovirus type 3 (PCV3) is a newly emerging circovirus that might be associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and cardiac and multisystemic inflammation. To aid the prevention and control of the infectious disease caused by PCV3, we developed a novel isothermal amplification assay using polymerase spiral reaction (PSR), which allows the visual detection of preserved strains and clinical samples.ResultsThis assay precisely amplified the PCV3 genome with the use of a water bath at 62 °C for 50 min. The detection limit was found to be 1.13 × 102 copies/μL by gel electrophoresis or with the use of a visible dye (an indicator comprising phenol red and cresol red). No cross-reaction with other porcine infectious viruses was observed. The detection results for 23 PCV3-positive samples by PSR were in accordance with loop-mediated isothermal amplification (LAMP) assay. The detection rate of the PSR assay for PCV3 positivity of clinical samples was 68/97, which was higher than LAMP assay (67/97).ConclusionsThese results indicated that the PSR assay provides an accurate and rapid method for the detection of PCV3 with high sensitivity and specificity. It is particularly suited for use in a simple laboratory setting without a thermal cycler or gel electrophoresis equipment.

Rapid visual detection of diarrhoea-associated eaeA gene using polymerase spiral reaction (PSR) from clinical samples

Diarroeagenic Escherichia coli including enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) is associated with acute diarrhoea in children and young animals. The virulence is associated with attaching and effacing lesions encoded by eaeA gene which is considered as marker for EPEC and EHEC. Diagnosis of EPEC and/or EHEC is traditionally done by conventional PCR assay, which sometimes provide false-negative result and also costly as well as difficult to perform at field level. A novel isothermal polymerase spiral reaction (PSR) is developed for detection of eaeA gene in E. coli isolated from diarrhoeic pig, cattle, sheep and goat. Samples were collected from diarrhoeic animals for isolation and identification of the E. coli. eaeA gene was detected by conventional PCR and PSR assays. A total of 39 isolates were recorded positive for eaeA gene by conventional PCR, whereas 52 isolates were recorded positive for eaeA gene by PSR assay. PSR technique was 100 times more sensitive than conventional PCR. PSR techniques was found to be more sensitive, specific, cost effective, user friendly and reliable technique over conventional PCR. The PSR technique developed in the present study can be applied for screening of the clinical isolates for confirmation of EPEC and/or EHEC.

Rapid visual isothermal nucleic acid-based detection assay of Brucella species by polymerase spiral reaction.

The aim of this study was to develop polymerase spiral reaction (PSR) for rapid, sensitive and specific detection of Brucella sp. Polymerase spiral reaction assay was developed using specifically designed primers targeting the conserved multicopy IS711 gene of Brucella sp. The assay could be performed within 60min at an isothermal temperature of 64°C. The lower limit of detection of PSR was 11·8fg and conventional PCR was 1·18pg of Brucella abortus genomic DNA. Thus, PSR was found to be 100-fold more sensitive than conventional PCR and was comparable to real-time PCR. The specificity of PSR was tested with other non-Brucella bacteria and also with some bacterial and viral pathogens causing abortions. The assay was found to be specific as it did not detect any putative pathogens other than Brucella sp. Fifty-six clinical samples suspected for brucellosis (aborted fetal stomach content) were screened with PSR to validate the applicability of the test to detect Brucella DNA. The same samples were also screened with conventional PCR and real-time PCR. Of 56 samples, 25 samples were found to be positive with both PSR as well as real-time PCR, whereas only 20 samples were found positive with conventional PCR. The results of this study indicated that the PSR assay is a simple, rapid, sensitive and specific method for the detection of Brucella sp. that may improve diagnostic potential in clinical laboratories or can be used at diagnostic laboratories with minimal infrastructure. The PSR assay, because of its simplicity and low cost, can be preferred to other molecular methods in the diagnosis of infectious diseases.

Novel Polymerase Spiral Reaction (PSR) for rapid visual detection of Bovine Herpesvirus 1 genomic DNA from aborted bovine fetus and semen

Bovine herpesvirus-1 (BHV-1) is a major viral pathogen affecting bovines leading to various clinical manifestations and causes significant economic impediment in modern livestock production system. Rapid, accurate and sensitive detection of BHV-1 infection at frozen semen stations or at dairy herds remains a priority for control of BHV-1 spread to susceptible population. Polymerase Spiral Reaction (PSR), a novel addition in the gamut of isothermal techniques, has been successfully implemented in initial optimization for detection of BHV-1 genomic DNA and further validated in clinical samples. The developed PSR assay has been validated for detection of BHV-1 from bovine semen (n=99), a major source of transmission of BHV-1 from breeding bulls to susceptible dams in artificial insemination programs. The technique has also been used for screening of BHV-1 DNA from suspected aborted fetal tissues (n=25). The developed PSR technique is 100 fold more sensitive than conventional PCR and comparable to real-time PCR. The PSR technique has been successful in detecting 13 samples positive for BHV-1 DNA in bovine semen, 4 samples more than conventional PCR. The aborted fetal tissues were negative for presence of BHV-1 DNA. The presence of BHV-1 in bovine semen samples raises a pertinent concern for extensively screening of semen from breeding bulls before been used for artificial insemination process. PSR has all the attributes for becoming a method of choice for rapid, accurate and sensitive detection of BHV-1 DNA at frozen semen stations or at dairy herds in resource constrained settings.

Polymerase spiral reaction (PSR): a novel, visual isothermal amplification method for detection of canine parvovirus 2 genomic DNA.

Canine parvovirus-2 (CPV-2), which is ubiquitously distributed worldwide, causes severe and often fatal gastroenteritis in dogs. Accurate, differential and rapid diagnosis of canine parvoviral enteritis remains a challenge for clinicians. A recently developed isothermal amplification technique, polymerase spiral reaction (PSR), was optimized for the first time for a viral pathogen with reference recombinant plasmid standards from different CPV-2 antigenic variants (CPV-2, CPV-2a, CPV-2b and CPV-2c) and subsequently validated using clinical samples. Addition of chromogenic substrate SYBR Green I after the completion of the reaction resulted in bright green fluorescence in positive samples, while negative samples and a no-template control remained orange. These results were further substantiated through visualization of a laddering pattern of the PSR-amplified product in an agarose gel in positive cases and the absence of this pattern in no-template control and negative samples. The PSR assay was found to be highly specific, as it did not react with other putative canine pathogens (canine adenovirus 1 and canine distemper virus). The sensitivity of the newly developed PSR technique was compared with that of conventional PCR, real-time PCR and LAMP, using a serial tenfold dilution of canine parvovirus DNA. The detection limit of PSR was found to be at the femtogram level, which is comparable with that of real-time PCR and LAMP, which are ten times more sensitive than conventional PCR. The assay was validated using 90 clinical samples, of which 54 were found positive, while only 45 samples were positive in conventional PCR. This novel assay, which is fully compliant with the 'ASSURED' concept for disease diagnosis, provides a simple, rapid, specific, sensitive and cost-effective method for diagnosis of canine parvoviral enteritis in veterinary clinics.

Rapid Detection of Candida albicans by Polymerase Spiral Reaction Assay in Clinical Blood Samples.

Candida albicans is the most common human yeast pathogen which causes mucosal infections and invasive fungal diseases. Early detection of this pathogen is needed to guide preventative and therapeutic treatment. The aim of this study was to establish a polymerase spiral reaction (PSR) assay that rapidly and accurately detects C. albicans and to assess the clinical applicability of PSR-based diagnostic testing. Internal transcribed spacer 2 (ITS2), a region between 5.8S and 28S fungal ribosomal DNA, was used as the target sequence. Four primers were designed for amplification of ITS2 with the PSR method, which was evaluated using real time turbidity monitoring and visual detection using a pH indicator. Fourteen non-C. albicans yeast strains were negative for detection, which indicated the specificity of PSR assay was 100%. A 10-fold serial dilution of C. albicans genomic DNA was subjected to PSR and conventional polimerase chain reaction (PCR) to compare their sensitivities. The detection limit of PSR was 6.9 pg/μl within 1 h, 10-fold higher than that of PCR (69.0 pg/μl). Blood samples (n = 122) were collected from intensive care unit and hematological patients with proven or suspected C. albicans infection at two hospitals in Beijing, China. Both PSR assay and the culture method were used to analyze the samples. Of the 122 clinical samples, 34 were identified as positive by PSR. The result was consistent with those obtained by the culture method. In conclusion, a novel and effective C. albicans detection assay was developed that has a great potential for clinical screening and point-of-care testing.