Conventional Real-time Polymerase Chain Reaction Research Articles

Machine Learning-Assisted, Dual-Channel CRISPR/Cas12a Biosensor-In-Microdroplet for Amplification-Free Nucleic Acid Detection for Food Authenticity Testing.

The development of novel detection technology for meat species authenticity is imperative. Here, we developed a machine learning-supported, dual-channel biosensor-in-microdroplet platform for meat species authenticity detection named CC-drop (CRISPR/Cas12a digital single-molecule microdroplet biosensor). This strategy allowed us to quickly identify and analyze animal-derived components in foods. This biosensor was enabled by CRISPR/Cas12a-based fluorescence lighting-up detection, and the nucleic acid signals can be recognized by a Cas12a-crRNA binary complex to trigger the trans-cleavage of any by-stander reporter single-stranded (ss) DNA, in which nucleic acid signals can be converted and amplified to fluorescent readouts. The ultralocalized microdroplet reactor was constructed by reducing the reaction volume from up to picoliter to accommodate the aforementioned reaction to further enhance the sensitivity to even render an amplification-free nucleic acid detection. Moreover, we established a smartphone App coupled with a random forest machine learning model based on parameters such as area, fluorescent intensity, and counting number to ensure the accuracy of image recording and processing. The sample-to-result time was within 80 min. Importantly, the proposed biosensor was able to accurately detect the ND1 (pork-specific) and IL-2 (duck-specific) genes in deep processed meat-derived foods that usually had truncated DNA, and the results were more robust and practical than conventional real-time polymerase chain reaction after a side-by-side comparison. All in all, the proposed biosensor can be expected to be used for rapid food authenticity and other nucleic acid detections in the future.

Enhancing Conventional PCR for Detection of Erwinia amylovora

Polymerase chain reaction (PCR) methods, including conventional PCR (cPCR) and quantitative real-time PCR (qRT-PCR), with both plasmid- and chromosome-targeting primers, are currently the most reliable methods for detecting <i>Erwinia amylovora</i> due to their high sensitivity and specificity. Despite qRT-PCR’s quantitative advantage, cPCR remains an attractive method to detect this bacterium in initial screenings of suspected host plants, as it is cost-effective and does not require skilled personnel in well-equipped laboratories. This study aimed to significantly improve cPCR robustness via application of bovine serum albumin (BSA) as a PCR facilitator, with a modified EaF/R primer pair, as previously reported. Experiments have shown that simple supplementation with BSA (10 mg/ml) enhances cPCR reactions using templates such as genomic DNA, bacterial cells, and infected symptomless host organs, including immature apple fruits and seedlings, with EaF/R primers. The cPCR method described in this study is simple, specific, and reliable, and can be applied in routine assays to diagnose fire blight.

Molecular Models of Toxoplasma gondii in Humans and Animals.

Toxoplasma gondii (T. gondii) is an obligate intracellular, zoonotic protozoan parasite of interest to physicians and veterinarians with its highly complex structure. It is known to infect about one-third of the world's population. Since it is a zoonotic disease, it is necessary to keep the animal population under control in order to prevent human exposure. Many studies have been conducted on the detection of T. gondii and it has been determined that there are three clonal groups consisting of types 1, 2, 3. Developments in molecular studies have led to changes in the taxonomy and new developments in parasitic diseases. It has helped in diagnosis, treatment, development of antiparasitic drugs and research on resistance. They also provided research on vaccine studies, genetic typing and phylogenetics of parasitic diseases. Conventional polymerase chain reaction (PCR), real-time PCR and genotyping studies conducted today increase our knowledge about T. gondii. Methods such as B1, SAG1, SAG2, GRA1, 529-bp repeat element, OWP genes and 18S rRNAs are mostly used in PCR, and methods such as MS, MLST, PCR-RFLP, RAPD-PCR and HRM are used in genotyping. Toxoplasmosis is a disease that is within the framework of the concept of one health and must attract attention, has not yet been eradicated in the world and needs joint studies for humans, animals and ecosystems to be eradicated. This can only be possible by establishing interdisciplinary groups, conducting surveys and training.

DETECTION OF MYCOPLASMA DISPAR IN BOVINE RESPIRATORY DISEASE BY POLYMERASE CHAIN REACTION ASSAY IN SULAIMANIYAH CITY

This study was aimed to identification and detection of Mycoplasma dispar by conventional real-time polymerase chain reaction (PCR) assays followed by routine hematoxylin & eosin technique to further confirmation of Mycoplasma species related to the lesions observed in diseased calves. Ten samples of infected lung tissue were gathered and fixed in 10% neutral buffered formalin and handled for making a microscopic slide after confirming Mycoplasma dispar infection by PCR. Histopathological examination of the lung revealed two types of pneumonia; chronic suppurative pneumonia in 60% of the cases and 40% of diffuse interstitial pneumonia. Mycoplasma dispar was confirmed based on genetic analysis. Slemani/2018 field isolate showed 99.84% homologies with four reference isolates of Mycoplasma dispar in NCBI GenBank. Phylogenetic analysis indicated a close relation to Mycoplasma ovipneumoniae, Mycoplasma hyopneumoniae, and Mycoplasma flocculare, often isolated from sheep and goats. Therefore, further study is crucial to identify Iraqi livestock's circulating Mycoplasma species related to respiratory diseases.

The diagnostic performance evaluation of Panbio and STANDARD Q coronavirus disease 2019 antigen tests against real-time polymerase chain reaction in southern Ethiopia

The coronavirus disease 2019 (COVID-19) pandemic has created a public health crisis. This study aimed to evaluate the diagnostic performance of the Panbio and STANDARD Q COVID-19 antigen rapid diagnostic tests (RDTs) against the real-time polymerase chain reaction (RT-PCR) at one of the largest hospitals in southern Ethiopia. Nasopharyngeal samples, which were collected during the pandemic from individuals suspected of COVID-19 and stored at − 70 °C, were analyzed in June and July 2022. The performance of the Panbio COVID-19 antigen tests was evaluated in 200 randomly selected nasopharyngeal samples (100 positives and 100 negatives for severe acute respiratory syndrome 2 by RT-PCR). The STANDARD Q test was evaluated using 100 positive and 50 negative samples. The respective sensitivity, specificity, positive predictive value and negative predictive values were 88%, 99%, 98.9% and 89.2% for the Panbio test and 91%, 98%, 98.9% and 84.5%, for the STANDARD Q test. The kappa values were 0.87 for the Panbio and 0.86 for the STANDARD Q test. Based on the findings presented here, the RDTs could be utilized as an alternative to conventional RT-PCR when it is challenging to diagnose COVID-19 owing to a lack of time, skilled lab personnel, or suitable equipment or electricity.

Ov-RPA-CRISPR/Cas12a assay for the detection of Opisthorchis viverrini infection in field-collected human feces.

Opisthorchis viverrini infection is traditionally diagnosed using the Kato-Katz method and formalin ethyl-acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O.viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA-CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA-CRISPR/Cas assay to detect O.viverrini infection in field-collected human feces. To detect O.viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA-CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA-CRISPR/Cas12a assay for detecting O.viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA-CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods. The Ov-RPA-CRISPR/Cas12a assay exhibited high specificity for detecting O.viverrini DNA. On the basis of the detection limit, the assay could detect O.viverrini DNA at concentrations as low as 10-1ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1ng. To validate the Ov-RPA-CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O.viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA-CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections. The Ov-RPA-CRISPR/Cas12a assay developed in this study can successfully detect O.viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O.viverrini infection, marking an initial step in the development of point-of-care diagnosis.

Evaluation of the diagnostic assays detecting red sea bream iridovirus infection at different severity levels

Red sea bream iridovirus (RSIV) is a highly contagious viral infection that affects various fish species and poses a significant threat to the global aquaculture industry. Thus, accurate and timely diagnosis is paramount for sustainable management of fish health. This study rigorously evaluated the diagnostic efficacy of various polymerase chain reaction (PCR) assays, focusing on those recommended by the World Organization for Animal Health (WOAH) and the assays newly proposed by WOAH's Aquatic Animals Health Standards Commission. Specifically, this study assessed conventional PCR, nested PCR, modified 1-F/1-R, and real-time PCR assays using a 95% limit of detection (LoD95%), as well as diagnostic sensitivity (DSe) and specificity (DSp) tests across different RSIV severity grades (G0−G4). In previous studies, the LoD95% for the 1-F/1-R and 4-F/4-R conventional assays were 225.81 and 328.7 copies/reaction, respectively. The modified 1-F/1-R exhibited a lower LoD95% of 51.32 copies/reaction. Notably, the nested PCR had an LoD95% of 11.23 copies/reaction, and the real-time PCR assay had an LoD95% of 12.02 copies/reaction. The DSe varied across RSIV severity grades, especially in the lower G0−G2 grades. The nested PCR and modified 1-F/1-R assays displayed the highest DSe, making them particularly useful for early-stage screening and detection of asymptomatic carriers. In addition, the PCR assays did not cross-react with any other aquatic pathogens except RSIV. Our findings significantly advanced the diagnostic capabilities of RSIVD by suggesting that nested PCR and modified 1-F/1-R assays are particularly promising for early detection. We propose their inclusion in future WOAH guidelines for a more comprehensive diagnostic framework.

PREVALENCE OF BOVINE MASTITIS AND THE FIRST DETECTION OF MYCOPLASMA BOVIS IN VIETNAM’S DAIRY FARMS

Mastitis still remains an economic disease in dairy farms all over the world. This study aimed to report the prevalence of this disease in large-scale farms in Vietnam, the pathogens profile, and first report the detection of Mycoplasma bovis (M. bovis) by applying conventional culture and real-time PCR (polymerase chain reaction) methods. We examined 64,802 enrolled dairy cows from six farms in Vietnam, yielding 1,874 (2.9%) total cases of clinical mastitis. The majority of the 14 pathogens were identified. Klebsiella spp. accounted for 21.0% of the total isolates. Streptococcus uberis (S. uberis), Escherichia coli (E. coli), other Streptococcus spp, and Coagulase-Negative Staphylococci (CNS), followed at 12.6, 7.6, 6.0, and 5.5 percent, respectively. The rest pathogens include Pseudomonas spp., Enterococcus faecalis, Enterobacter spp., Bacillus spp., Citrobacter spp., Pasteurella spp., Proteus spp., and Staphylococcus aureus were isolated with less than 1% each. To the best of our knowledge, this is the first report for detecting M. bovis in mastitis milk samples in Vietnam, where 35 out of 1,422 samples submitted in 2022 were M. bovis positive. Moreover, of the 35 positive cases, 11 cases had only M. bovis, while 24 cases had M. bovis and other pathogens, including 13 Enterobacteriaceae, 7 Streptococcus spp., and 4 CNS.

Evaluation of loop-mediated isothermal amplification method for efficient detection of the periodontopathic bacteria Porphyromonas gingivalis.

Periodontitis is a multifactorial, polymicrobial oral inflammatory illness brought on by oral pathogens. Porphyromonas gingivalis is a Gram-negative, obligatory anaerobic, black-pigmented coccobacillus and is regarded as a primary etiological factor in the progression of periodontitis. Rapid, highly senstitive and specific detection methods are emerging. The present study aimed to evaluate the loop-mediated isothermal amplification (LAMP) technique for efficiently detecting P. gingivalis from subgingival plaque samples of chronic periodontitis patients. This study included 50 subgingival plaque samples from patients suffering from chronic periodontitis. The DNA (Deoxyribonucleic acid) was extracted by the "modified proteinase K" method. A set of six primers, targeting the pepO gene of P. gingivalis, was used for conducting LAMP. The amplification was visualized by naked-eye detection and agarose electrophoresis. Conventional polymerase chain reaction (PCR) and real-time qantitative PCR (qPCR) were carried out by targeting the 16SrRNA (16S ribosomal ribonucleic acid) gene of P. gingivalis. The results showed that LAMP detected P. gingivalis in 40 out of 50 samples (80%). Whereas, qPCR and conventional PCR technique detected P. gingivalis in 38 (76%) and 33 (66%) samples respectively. The sensitivity and specificity of the LAMP method were 94.87% and 90.90%, respectively. With qPCR, the sensitivity and specificity were found to be 92.30% and 81.81%, respectively, whereas, with conventional PCR, it was found to be 76.92% and 72.72%, respectively. LAMP is an efficient technique for quick, accurate, and reliable identification of P. gingivalis from subgingival plaque samples. The technique needs to be validated analytically, and further studies can be conducted by taking saliva and/or gingival crevicular fluid samples from periodontitis patients.

Comparison of Serological and Molecular Methods for Detection of Spiroplasma citri in Moroccan Citrus-Growing Areas.

Spiroplasma citri, a helical motile, wall-less, and cultivable microorganism of the class Mollicutes, is the agent of the citrus stubborn disease. There is currently a lack of data about the presence of this pathogen in Moroccan citrus orchards. This study aims to validate serological and molecular methods for routine S. citri diagnosis in Moroccan citrus groves. To provide an update on the present status of the outbreak of the pathogen in Moroccan citrus orchards, a survey of S. citri was conducted in the main citrus-growing regions of Morocco. A total of 575 leaf samples were collected from citrus trees with symptoms attributable to S. citri infection. Samples were collected during 2020 and 2021 from 23 citrus orchards. The presence of S. citri was tested in all samples using the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Using this method, 57 samples were found to be infected with S. citri, 41 samples had doubtful results, and the remaining samples were negative. To corroborate the results of the DAS-ELISA test, 148 samples were chosen for additional molecular testing using conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) based on specific primer pairs targeting three different genes (putative adhesion-like gene P58, putative adhesion gene P89, and spiralin gene). Using primers that target the putative adhesion-like gene P58, S. citri was detected by conventional and real-time PCR amplification from plant tissue with differing degrees of specificity. The results allowed us to determine the incidence of S. citri in all Moroccan citrus orchards, with a wide range of positive samples varying from 6.5% to 78%, and to show that molecular tests, particularly real-time PCR assays that target the putative adhesion-like gene P58, are the most sensitive for making an accurate diagnosis of S. citri. Indeed, the real-time PCR with P58-targeting primers yielded positive results from all positive and doubtful ELISA samples as well as some negative samples, with an OD value close to 1.5× times healthy samples, thus demonstrating a high sensibility of this technique.

Identification and characterisation of lumpy skin disease virus recently isolated from Giza, Egypt

Lumpy skin disease (LSD) is a viral disease, geographically distributed in Africa and now, vigorously spread in the Near East and also in Europe and Asia. It has a significant economic impact on cattle industry in Africa. The aim of this study was isolation and rapid identification of LSD virus circula­ting in Egypt from clinically suspected cattle based on clinical and molecular basis in a rapid and accurate way. Fifteen representative specimens (skin sitfasts) were collected in 2018 from clinically infected cattle in Giza governorate, Egypt. The virus was isolated on chorioallantoic membrane of specific pathogen free embryonated chicken eggs and Madin Darby Bovine Kidney tissue culture cells. The isolated virus was identified and confirmed by conventional polymerase chain reaction (PCR) and real-time PCR. Histopathological examination of the lesions showed a pathognomic intracytoplasmic inclusion body in dermal stroma section. The section of dermal layer revealed vasculitis with projection of its endothelial lining. It was concluded that LSD was enzootic in Egypt and still circulating among Egyptian cattle so that LSD virus could be isolated and identified by traditional and molecular diagnostic methodes. Real time PCR assay could be applied for rapid and accurate confirmation of the field isolate of LSD virus.

Epidemiologic Study of Diarrhea Pathogens Detected by Multiplex Real-Time PCR: a Single Center Study During 1 Year.

Acute gastroenteritis is one of the major causes of morbidity and mortality worldwide, especially in children and the elderly. The identification of various diarrhea-causing bacteria using multiplex polymerase chain reaction (PCR) and rapid antigen testing has enabled a more detailed analysis of diarrhea-causing pathogens. Pre-vious reports have a limitation in that they do not include data on multiple infections in which two or more infectious agents are simultaneously detected, and there are no data on clinical information. We investigated various diarrhea-causing bacteria and viruses detected by multiplex real-time PCR for one year at a single institution. This study included 766 subjects who underwent multiplex real-time PCR testing of direct stool specimens for the purpose of diagnosis from April 2019 to February 2020. The multiplex PCR test used in our study can simultaneously detect 16 types of bacteria and five types of viruses. When two or more pathogens were detected by multiplex real-time PCR, they were confirmed using single conventional PCR or real-time PCR. Demographic, clinical, and laboratory data were collected from electronic medical records (EMR). The detected bacteria and viruses were analyzed according to age and season. Out of a total of 352 stool samples with pathogen detection, 265 (75.3%) were detected as single and 87 (24.7%) showed co-detection. The highest rates of single and co-detection were for Clostridium perfringens, and the highest combination of co-infections was for C. perfringens and Staphylococcus aureus. We demonstrated that different age groups showed varying pathogen distributions. While no special seasonality was found in the monthly distribution, it should be noted that the total number of cases peaked in September. The data presented in our study serves as epidemiologically important basic data.

Diagnosis of Pebrine Disease in Silkworm Using Molecular Methods.

Since pebrine disease, as the most important and dangerous disease in silkworms, spreads horizontally through the spores and vertically through the eggs, combating the disease and eliminating it completely from livestock production has been associated with numerous problems. This project aimed to identify the molecular cause of pebrine disease in silkworms using a sensitive, specific, and accurate method. To this purpose, a 136 bp fragment was selected based on the Nosema bombycis partial SSU rDNA sequence, and a pair of primers was designed. Afterward, using the conventional polymerase chain reaction (PCR) method, the target fragment was amplified and sequenced. After that, to determine the detection sensitivity, using the Real-Time PCR method, 5-fold serial dilutions of N. bombycis DNA were prepared, and the last dilution that produced a fluorescent signal was considered the minimum detection limit. All tests were performed in duplicates. Based on the results of the sensitivity test, the standard curve including Ct values ​​and DNA concentration was used for analysis. Moreover, 80 unknown samples examined by light microscope were evaluated using conventional PCR and Real-Time PCR. Both PCR results showed no amplification for the negative control samples. The findings demonstrated that the lowest detection limit for N. bombycis was less than 6 pg of DNA, while, this amount was 8 ng for conventional PCR. Out of 80 samples examined, 55, 60, and 62 samples were positive for light microscope, conventional PCR, and Real-Time PCR methods, respectively. The findings suggested that the Real-Time PCR method had a higher ability to detect the causative agent of pebrine disease than the conventional PCR method, and both methods were superior to light microscopy. Therefore, due to the fewer steps and higher accuracy of Real-Time PCR, it can be introduced as a suitable method for diagnosing pebrine disease.

Detection of EhCRT Gene Expression in Entamoeba histolytica-Infected Children and its Correlation with Interleukin 25 and Tumor Necrosis Factor Alpha

Objectives: Entamoeba histolytica is a human enteric protozoan, which is the causative agent of amebiasis. The host activates a series of immunological responses to protect against the parasite after contact with the ameba and further invasion of the gut epithelium layer. As a result, the ameba has developed a variety of evasion mechanisms to hold out the immune response and continue to survive and cause disease. The calreticulin (EhCRT) is one of the immunogenic molecules of E. histolytica that induces an immune response in the human host. Increase in the expression of the EhCRT gene could provide control mechanism that allows the parasite to adapt and survive in host tissues. Aim of the Study: This study was designed to detect the EhCRT gene of E. histolytica by real-time polymerase chain reaction (PCR) in stool samples of children with amebiasis and its roles in host–parasite relationship via measuring the concentration of tumor necrosis factor alpha (TNFα) and interleukin 25 (IL25) by enzyme-linked immunoassay (ELISA) technique in their serum. Materials and Methods: A total of 86 diarrheal fecal samples were collected from children in age <1 year to 13 years suspected to be infected with E. histolytica during the period from December 30, 2020, to September 1, 2021. Microscopically positive samples were the subject to conventional PCR and real-time PCR for the detection of E. histolytica HM1:IMSS strain using (Psp) gene sequences and detection of calreticulin (EhCRT) expression. Blood was withdrawn from each child included in the study for ELISA test to measure the level of IL25 and TNFα. Results: Fecal samples for microscopic examination revealed that 71 (82.6%) children had amebic colitis, E. histolytica gene was detected in 44 samples (71%) using conventional PCR, and the immunogene EhCRT was expressed in 36 stool samples using real-time PCR. The results of the recent study showed highly significant elevation in the level of TNFα and IL25 in the amebic group (Eh+ve PCR). The majority of amebic children were in the age group of 1–4 years, had mucoid, acute, and with primary episodes of diarrhea. Conclusion: E. histolytica is a protozoan parasite highly prevalent among diarrheal children and is responsible for gastrointestinal amebiasis in the human host. The PCR is a useful tool in the diagnosis of E. histolytica infection. It is clear that the expression of the calreticulin gene (EhCRT) concedes with the duration of diarrhea a virulence factor that plays a role in host pathogenic pathways. The findings of this study showed that the level of TNFα in the serum of children infected with amebic colitis (Eh gene + ve) is significantly increased during the course of infection and the cytokine IL25 exhibits a significant drops in the same children.

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.

Detection of Streptococcus suis using the optimized real-time polymerase chain reaction protocol

The article presents the results of studies on the detection of Streptococcus suis by real-time polymerase chain reaction. Isolation and species identification of the studied isolates of streptococci was carried out according to morphological, cultural, biochemical and biological properties by conventional methods. The study of cultural characteristics of growth was carried out using conventional bacteriological methods on the brain heart infusion broth (BHI) and BHI agar with the addition of 5% sheep blood (blood BHI agar). To confirm biochemical properties as a confirmatory method, API 20 STREP test kit (bioMerieux, France) was used. In addition, to differentiate S. suis from the non-pathogenic species of streptococci, the hemolysis test was used. As a result of the studies, it was found that the use of the real-time PCR (polymerase chain reaction) method makes it possible to detect S. suis in an amount of 1 x 104 genome copies in the sample. All described validation parameters for the qualitative detection of S. suis DNA by real-time PCR meet international requirements, which guarantees accurate and reliable results. In Ukraine only a diagnostic test kit for convential PCR has been developed for the detection of swine streptococcosis. This approach is more time consuming and complex in comparison with the real-time PCR approach. We recommend that diagnostic laboratories implement this method in their practice. This will increase the number of effective diagnostic tools available to veterinarians on pig farms when they order laboratory tests. The high analytical sensitivity limit of a test is an essential parameter when screening is the focus, and obtaining false negative results causes a risk of the development of infection process among pig populations within infected herds. Our study showed that microbiological diagnostic methods to determine morphological and cultural properties can identify S. suis at the genus level. Determination of biochemical properties using the API 20 STREP test kit can be used to identify S. suis 1 and 2 serotypes. The conventional method and real-time PCR have 100% specificity and can be used to identify S. suis of different serotypes. Real-time PCR is a 2 to 4 times more sensitive limit than conventional PCR depending on the serotype being studied, and can be used to more accurately identify streptococcal DNA. It was found that the use of the real-time PCR method makes it possible to detect S. suis in an amount of 1 x 104 copies of the genome in the sample. Additionally, it was found that all the studied validation parameters of the qualitative method for determining S. suis DNA by real-time PCR meet international requirements, which guarantees accurate and reliable results.

Performance of Molecular Tests in the Diagnosis of Syphilis From 2009 to 2019: A Systematic Review and Meta-Analysis.

Syphilis continues to be a public health problem, and its diagnosis still has limitations. Molecular diagnosis provides an alternative for rapid and effective management. The objective is to determine the accuracy of tests in the molecular diagnosis of syphilis. We searched PubMed and Web of Sciences for articles related to molecular detection of syphilis from January 1, 2009, to December 31, 2019. The bivariate Reitsma model and the hierarchical receiver operating characteristic curve model were used to evaluate the diagnostic performance of molecular tests at a 95% confidence interval. A subgroup meta-analysis was performed to explore sources of heterogeneity. Forty-seven articles were identified for qualitative synthesis, of which 23 met the inclusion criteria for meta-analysis. The pooled sensitivities in conventional polymerase chain reaction (PCR) and real-time PCR were 77.52 (59.50-89.01) and 68.43 (54.96-79.39), respectively. The pooled specificities were 98.00 (90.73-99.59) and 98.84 (97.55-99.46), respectively. Ulcer samples had a better performance (sensitivity of 79.88 [69.00-87.62] and specificity of 98.58 [97.25-99.27]), and the major target genes were the polymerase A gene and tpp47 gene. Our work showed that conventional PCR was more widely used than real-time PCR in the diagnosis of syphilis, and ulcers were the best specimens. Sample types and target genes are factors that may influence the quality of the different tests. These results could provide evidence for further work in the direction of providing a more efficient diagnostic test.

Development of an Accurate and Sensitive Diagnostic System Based on Conventional PCR for Detection of African Swine Fever Virus in Food Waste

African swine fever virus (ASFV), a highly contagious virus, can cause diseases with high mortality rates in pigs, making it a pathogen of social and economic significance. ASFV has been reported to show potential long-term survival in living livestock, such as pigs, but also in leftover cooking meat and undercooked pork meat. Hence, it is possible that there could be direct reinfection or secondary infection through feed produced from household food waste and treatment facilities. Many polymerase chain reaction (PCR)-based molecular diagnostic techniques to detect ASFV in clinical swine samples have been reported. However, those with applicability for food waste samples, which contain relatively low viral copy numbers and may contain various unknown inhibitors of PCR, are still lacking. In this study, we developed a conventional PCR-based diagnostic system that can detect ASFV with high sensitivity from food waste sample types. The technique shows a 10–100 times higher limit of detection compared to that of previously reported methods based on conventional PCR and quantitative real-time PCR. It is also capable of amplifying a sequence that is approximately 751 nucleotides, which is advantageous for similarity analysis and genotyping. Moreover, a ASFV-modified positive material different from ASFV that could synthesize 1400 nucleotide amplicons was developed to identify false-positive cases and thus enhance diagnostic accuracy. The method developed herein may be applicable for future ASFV monitoring, identification, and genotyping in food waste samples.

Review on Molecular Diagnosis of Cestode and Metacestode in Cattle

Cestode infestations in animals are the most important parasite of livestock and humans because most of these parasites are zoonotic causing cysticercosis and hydatidosis in man and it causes economic and production losses in livestock. Diagnosis of Taenia Spp by microscopic observation lack sensitivity and specificity and detection by enzyme-linked immunosorbent assay (ELISA) technique form cross-reaction. The molecular diagnostic can be best to detect in adult and larval stage in definitive and intermediate host based on the amplification of deoxyribonucleic acid (DNA) of target gene with the primer using a different technique of polymerase chain reaction (PCR) such as multiplex PCR. Conventional PCR, real-time PCR, nested PCR, and PCR-restriction fragment length polymorphism (RFLP) are highly sensitive for the diagnosis of cestode and metacestode. Those diagnoses are used for differentiation of Taenia species and differentiation of Taenia and Echinococcus species. As compared to other diagnostic techniques most molecular methods have higher sensitivity and specificity but due to the relatively higher cost, few are commercially available. Most of the molecular diagnostic tests developed to date are generally applicable for laboratory research purposes. The developments in the genomic and proteomic analysis should be used for further understanding of parasite-animal host interaction to find additional targets for diagnosis.

Usefulness of the FilmArray Meningitis/Encephalitis Panel in diagnosis of central nervous system infection after allogeneic hematopoietic stem cell transplantation.

The BioFire FilmArray® Meningitis/Encephalitis Panel (FAMEP) is designed to rapidly and accurately detect common multiple pathogens that cause central nervous system (CNS) infection, including viruses, bacteria, and yeast. The FAMEP's usefulness in the setting of allogeneic hematopoietic stem cell transplantation (HSCT) has not been fully evaluated. This retrospective study evaluated the usefulness of the FAMEP in the screening for CNS infection after allogeneic HSCT. Cerebrospinal fluid (CSF) was obtained from 12 patients to evaluate the causes of CNS disorders after allogeneic HSCT, and the FAMEP was applied. The median day of the FAMEP evaluations was 27days post-transplant (range, 0-390). Human herpesvirus 6 (HHV-6) was detected in three patients and cytomegalovirus was detected in one patient, leading to the diagnosis of encephalitis/myelitis. In three patients (HHV-6, n = 2; CMV, n = 1), the presence of the viruses was confirmed by conventional real-time polymerase chain reaction (PCR). However, in the remaining patient with HHV-6 detected by the AMEP, HHV-6 was not detected by real-time PCR at the onset but was detected 7days later. The treatments for the detected viruses improved the clinical conditions in the four patients. Our results suggest that the FAMEP can be a useful sensitive assay in the screening and diagnosis of CNS viral infections after allogeneic HSCT.