Diagnosis model of early Pneumocystis jirovecii pneumonia based on convolutional neural network: a comparison with traditional PCR diagnostic method.

BACKGROUND: Pneumocystis jirovecii pneumonia (PJP) is an interstitial pneumonia caused by pneumocystis jirovecii (PJ). The diagnosis of PJP primarily relies on the detection of the pathogen from lower respiratory tract specimens. However, it faces challenges such as difficulty in obtaining specimens and low detection rates. In the clinical diagnosis process, it is necessary to combine clinical symptoms, serological test results, chest Computed tomography (CT) images, molecular biology techniques, and metagenomics next-generation sequencing (mNGS) for comprehensive analysis. PURPOSE: This study aims to overcome the limitations of traditional PJP diagnosis methods and develop a non-invasive, efficient, and accurate diagnostic approach for PJP. By using this method, patients can receive early diagnosis and treatment, effectively improving their prognosis. METHODS: We constructed an intelligent diagnostic model for PJP based on the different Convolutional Neural Networks. Firstly, we used the Convolutional Neural Network to extract CT image features from patients. Then, we fused the CT image features with clinical information features using a feature fusion function. Finally, the fused features were input into the classification network to obtain the patient's diagnosis result. RESULTS: In this study, for the diagnosis of PJP, the accuracy of the traditional PCR diagnostic method is 77.58%, while the mean accuracy of the optimal diagnostic model based on convolutional neural networks is 88.90%. CONCLUSION: The accuracy of the diagnostic method proposed in this paper is 11.32% higher than that of the traditional PCR diagnostic method. The method proposed in this paper is an efficient, accurate, and non-invasive early diagnosis approach for PJP.

A semi-nested PCR method with increased sensitivity for the specific, direct detection of Salmonella enterica strains in poultry ectoparasites.

The darkling beetle, Alphitobius diaperinus, and the poultry red mite, Dermanysuss gallinae are among the most common pests of poultry farms. Both pests can be carriers and reservoirs of various pathogens including zoonotic ones like Salmonella. Salmonellosis is one of the most common foodborne diseases reported in the EU. We developed a semi-nested PCR method for the direct detection of Salmonella enterica. When testing the specificity of the novel PCR, we successfully detected various S. enterica strains, whereas Escherichia coli and Citrobacter strains gave negative results. The authenticity of the PCR products was confirmed by DNA sequencing. The sensitivity of the semi-nested PCR was tested on serial dilution of bacterial cultures and extracted DNA. We found our new method more sensitive than the previous PCRs. We also screened ectoparasite samples, collected from a poultry farm in Hungary, and three out of the eight samples were positive for S. Enteritidis. This novel PCR seems suitable for the detection of S. enterica strains in poultry ectoparasites without the need of sample pre-enrichment.

Development of a panel for detection of pathogens in xenotransplantation donor pigs.

There have been high expectations in recent years of using xenotransplantation and regenerative medicine to treat humans, and pigs have been utilized as the donor model. Pigs used for these clinical applications must be microbiologically safe, that is, free of infectious pathogens, to prevent infections not only in livestock, but also in humans. Currently, however, the full spectrum of pathogens that can infect to the human host or cause disease in transplanted porcine organs/cells has not been fully defined. In the present study, we thus aimed to develop a larger panel for the detection of pathogens that could potentially infect xenotransplantation donor pigs. Our newly developed panel, which consisted of 76 highly sensitive PCR detection assays, was able to detect 41 viruses, 1 protozoa, and a broad range of bacteria (by use of universal 16S rRNA primers). The applicability of this panel was validated using blood samples from uterectomy-born piglets, and pathogens suspected to be vertically transmitted from sows to piglets were successfully detected. We estimate that, at least for viruses and bacteria, the number of target pathogens detected by the developed screening panel should suffice to meet the microbiological safety levels required worldwide for xenotransplantation and/or regenerative therapy. This panel provides greater diagnosis options to produce donor pigs so that it would render unnecessary to screen for all pathogens listed. Instead, the new panel could be utilized to detect only required pathogens within a given geographic range where the donor pigs for xenotransplantation have been and/or are being developed.

Q fever and toxoplasmosis in South African livestock and wildlife: a retrospective study on seropositivity, sporadic abortion, and stillbirth cases in livestock caused by Coxiella burnetii.

BACKGROUND: Q fever and toxoplasmosis are economically important zoonoses as they cause considerable losses in livestock (cattle, sheep and goats) and wildlife (antelopes, giraffes, lions, and cheetahs) through reproductive disorders such as abortions and stillbirths. Q fever and toxoplasmosis testing in South Africa is conducted by the Agricultural Research Council-Onderstepoort Veterinary Research (ARC-OVR). However, both zoonoses are understudied and not monitored in South Africa as they are not considered controlled or notifiable diseases in the Animal Disease Act 35 of 1984. A retrospective study was conducted on Q fever (2007-2009) and toxoplasmosis (2007-2017) using diagnostic laboratory data at the ARC-OVR. Also, we report on sporadic abortion and stillbirth cases in livestock from diagnostic tissue samples submitted for Coxiella burnetii polymerase chain reaction (PCR) detection at the ARC-OVR. RESULTS: During 2007 to 2009, 766 animal samples were tested for C. burnetii antibodies and seropositivity was 0.9% (95%CI: 0.3-1.7) with sheep (1.9%; 95%CI: 0.6-4.4) having the highest seropositivity followed by cattle (0.7%; 95%CI: 0.09-2.6), while all goats (0.0%; 95%CI: 0.0-4.2) and wildlife (0.0%; 95%CI: 0.0-2.5) tested were negative. From 2007 to 2017, 567 sera were tested for T. gondii antibodies; overall seropositivity was 12.2% (95%CI: 9.6-15). Wildlife had highest seropositivity to T. gondii antibodies (13.9%; 95%CI: 9.0-19.7) followed by goats (12.9%; 95%CI: 9.2-17.4) and sheep (12.3%; 95%CI: 5.1-23.8) while seropositivity in cattle was 2.4% (95%CI: 0.06-12.9). Of 11 animals tested by C. burnetii PCR detection (2021-2022), 10 (91.0%) were positive. The amplicon sequences showed similarity to Coxiella burnetii strain 54T1 transposase gene partial coding sequence. CONCLUSIONS: We have confirmed the occurrence of the causative agents of Q fever and toxoplasmosis in livestock and wildlife in South Africa, with data limitations. These zoonoses remain of importance with limited information about them in South Africa. This study provides baseline information for future studies on Q fever and toxoplasmosis in South African livestock and wildlife, as well other African countries. Due to limited data collection experienced in this study, it is recommended that improvements in data collection samples tested should include associated factors such as sex, age, and breed of the animals.

Detection of the pathogenic fungus Cordyceps farinosa in the Thitarodes armoricanus soil-rearing environment based on nucleic acid targets.

Cordyceps farinosa, an entomopathogenic fungus, infects and leads to high mortality of Thitarodes armoricanus larvae, which die soon after the infection of C. farinose, usually before the colonization of Ophiocordyceps sinensis owing to competitive inhibition and fruiting body formation. Therefore, monitoring C. farinosa in the O. sinensis cultivation environment is critical for minimizing the C. farinosa infection-induced losses. In this study, we initially designed a PCR primer pair (Tar-1F/Tar-1R) through open reading frame prediction and homology comparison of the C. farinosa genome sequence. This primer pair can detect both C. farinosa and Samsoniella hepiali. To further distinguish, primers (ITS5-172/ITS4-95) were then designed to selectively amplify the large ribosomal subunit sequences in the C. farinosa genome. All these primers were applied in combination for detection of C. farinosa in soil samples. The sensitivity reached a detection limit of 1 × 106 spores/g soil. In addition, these primers can detect the presence of C. farinosa in dead T. armoricanus larval samples. This newly established rapid detection method provides important information for C. farinosa control during O. sinensis cultivation.

Epidemiological and genetic characteristics of porcine circovirus 3 in 15 provinces and municipalities of China between 2016 and 2020.

Porcine circovirus 3 (PCV3) is a newly emerging virus and has been found associated with porcine dermatitis and nephropathy syndrome in pigs. Compared with PCV2, research into PCV3 cap gene sequencing is deficient. To investigate the prevalence and genotype distribution of PCV3, we collected 1291 samples from 211 pig farms throughout 15 provinces and municipalities. 312 out of 1291 samples were tested positive by PCR. We further sequenced and analyzed 164 PCR-positive samples. The majority (61.8%) of isolates we sequenced belong to genotype PCV3c. PCV3c is also the dominant genotype in Hubei, Hunan, Hebei province and Chongqing city. We found 3 sites under positive selection and located in predicted epitope peptide, revealing that the pig's immunity may be a reason those sites are undergoing highly positive selection.

Molecular investigation of the incidence of Candida auris infections at selected hospitals in Iran.

BACKGROUND: The accurate occurrence rate of C. auris infections is still not clear, mainly due to the defects in detection and identification tools routinely used. In this study, we used conventional PCR and real-time PCR assays for sensitive and specific detection/identification of C. auris from either yeast isolates or clinical specimens collected from various patients in different parts of Iran. Our survey is the first large-scale study rating the incidence of C. auris infections in Iran. METHODS: A total of 439 yeast isolates and 590 clinical specimens were screened by specific C. auris-PCR, targeting the ITS region. The validity of positive samples was assessed by sequencing. RESULTS: Four out of 590 clinical specimens (0.68%) were positive by conventional PCR, while in real-time PCR performed on 100 clinical samples, including those four samples positive in conventional samples, 6 samples were positive. A complete agreement of the identification of positive cases with sequencing results was documented. Among 439 culture isolates, none was positive for C. auris. After following up and resampling of the patients with positive PCR, only one specimen showed positive culture for C. auris, which was confirmed by sequencing. CONCLUSION: C. auris is not a common cause of systemic or superficial fungal infections in Iran, and a few detected positive cases can be considered as a commensal, coloniser or infecting yeast which may potentially emerge in some clinical and therapeutical conditions. Mycological and phenotypical assays are not sensitive approaches for isolation/identification of C. auris, unless a specific and sensitive molecular-based method is applied.

Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry.

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

NGS analysis revealed that Lactococcus garvieae Lg-Per was Lactococcus petauri in Türkiye.

Lactococcus garvieae Lg-per was originally isolated from rainbow trout cultured in cages located on the Turkish coast of the Black Sea in 2011. A whole genome sequence of Lg-per was performed in the present study. The complete genome of Lg-per mapped to the reference genomes of L. garvieae (GCF_000269925.1) and Lactococcus petauri (GCF_014830225.1) had a total of 1,694,407 and 1,945,297 base pairs, respectively. Lg-per had 1955 protein-coding genes and 4 rRNA, 46 tRNA and 1 tmRNA operons. The orthoANI value was 98.30% between Lg-per and L. petauri (GCF_014830225.1) and 93.1% between Lg-per and L. garvieae (GCF_000269925.1). A phylogenetic tree generated from the whole genome sequences (WGS) of several Lactococcus species found that L. petauri (GCA 002154895) was closely related to the Lg-per strain with 98% similarity. Although L. garvieae Lg-per was confirmed as L. garvieae based on phenotypical, biochemical and 16S rRNA sequence, WGS of the Lg-per strain revealed that Lg-per was L. petauri. Using a 16S rRNA-based PCR detection approach, Lg-per was misdiagnosed as L. garvieae since its 16S rRNA gene was 99.9% similar to that of L. garvieae strains. Consequently, the 16S rRNA-based PCR detection approach may not be adequate for the identification of the Lactococcus genus. This is the first study to document the presence of L. petauri in Türkiye. L. garvieae isolates should be analysed using WGS since the same issue might occur in other countries.

Staphylococcal Enterotoxin Genes in Coagulase-Negative Staphylococci-Stability, Expression, and Genomic Context.

In the current study, we screened a collection of coagulase-negative staphylococci (CoNS) isolates for orthologues of staphylococcal enterotoxins (SEs) involved in S. aureus-related staphylococcal food poisoning (SFP). The amplicons corresponding to SEs were detected in S. chromogenes, S. epidermidis, S. haemolyticus, S. borealis, S. pasteuri, S. saprophyticus, S. vitulinus, S. warneri, and S. xylosus. All amplicons were sequenced and identified as parts of known S. aureus or S. epidermidis SE genes. Quantitative real-time PCR allowed determining the relative copy number of each SE amplicon. A significant portion of the amplicons of the sea, seb, sec, and seh genes occurred at low copy numbers. Only the amplicons of the sec gene identified in three isolates of S. epidermidis displayed relative copy numbers comparable to sec in the reference enterotoxigenic S. aureus and S. epidermidis strains. Consecutive passages in microbiological media of selected CoNS isolates carrying low copy numbers of sea, seb, sec, and seh genes resulted in a decrease of gene copy number. S. epidermidis isolates harbored a high copy number of sec, which remained stable over the passages. We demonstrated that enterotoxin genes may occur at highly variable copy numbers in CoNS. However, we could identify enterotoxin genes only in whole-genome sequences of CoNS carrying them in a stable form at high copy numbers. Only those enterotoxins were expressed at the protein level. Our results indicate that PCR-based detection of enterotoxin genes in CoNS should always require an additional control, like analysis of their presence in the bacterial genome. We also demonstrate S. epidermidis as a CoNS species harboring SE genes in a stable form at a specific chromosome site and expressing them as a protein.

Plasmodium cynomolgi Co-infections among Symptomatic Malaria Patients, Thailand.

Among 1,180 symptomatic malaria patients, 9 (0.76%) infected with Plasmodium cynomolgi were co-infected with P. vivax (n = 7), P. falciparum (n = 1), or P. vivax and P. knowlesi (n = 1). Patients were from Tak, Chanthaburi, Ubon Ratchathani, Yala, and Narathiwat Provinces, suggesting P. cynomolgi is widespread in this country.

Comparison of the CapitalBio™Mycobacterium RT-PCR detection test and Xpert MTB/RIF assay for diagnosis of renal tuberculosis.

The purpose of this study is to compare the efficiency difference between CapitalBio™Mycobacterium real-time polymerase chain reaction (RT-PCR) detection test and Xpert MTB/RIF assay for the diagnosis of renal tuberculosis (TB). We analyzed 117 samples collected between July 1, 2018, and October 31, 2019, from patients with suspected renal TB to determine the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the curve (AUC) of the CapitalBio™ Mycobacterium RT-PCR detection test for renal TB and to evaluate its diagnostic accuracy compared with Xpert MTB/RIF assay. Five cases were excluded from this study because of incomplete data. Taking clinical diagnosis as the gold standard, for the Xpert MTB/RIF assay, the sensitivity was 87.95% (78.96-94.07%), specificity 96.55% (82.24-99.91%), PPV 98.65% (92.70-99.97%), NPV 73.68% (56.90-86.60%), and AUC 0.92 (0.86-0.96). For the CapitalBio™Mycobacterium RT-PCR detection test, the overall sensitivity was 84.34% (74.71-91.39%), specificity 93.10% (77.23-99.15%), PPV 97.22% (90.32-99.66%), NPV 67.50% (50.87-81.43%), and AUC 0.89(0.81-0.94). The diagnostic efficiency of the CapitalBio™Mycobacterium RT-PCR detection test was similar to that of the Xpert MTB/RIF assay in patients with renal TB. Hence, the CapitalBio™Mycobacterium RT-PCR detection test presents a valuable alternative for the diagnosis of renal TB.

Detection and infectivity of SARS-CoV-2 in exhumated corpses.

Postmortem detection of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) after the exhumation of a corpse can become important, e.g. in the case of subsequent medical malpractice allegations. To date, data on possible detection periods [e.g. by reverse transcription polymerase chain reaction (RT-PCR)] or on the potential infectivity of the virus after an exhumation are rare. In the present study, these parameters were examined in two cases with a time span of approximately 4 months between day of death and exhumation. Using SARS-CoV-2 RT-PCR on swabs of both lungs and the oropharynx detection was possible with cycle threshold (Ct) values of about 30 despite signs of beginning decay. RT-PCR testing of perioral and perinasal swabs and swabs collected from the inside of the body bag, taken to estimate the risk of infection of those involved in the exhumation, was negative. Cell culture-based infectivity testing was negative for both, lung and oropharyngeal swabs. In one case, RT-PCR testing at the day of death of an oropharyngeal swab showed almost identical Ct values as postmortem testing of an oropharyngeal swab, impressively demonstrating the stability of viral RNA in the intact corpse. However, favorable climatic conditions in the grave have to be taken into account, as it was wintertime with constant low temperatures. Nevertheless, it was possible to demonstrate successful postmortem detection of SARS-CoV-2 infection following exhumation even after months in an earth grave.

Conventional PCR assisted single-component assembly of spherical nucleic acids for simple colorimetric detection of SARS-CoV-2.

Continuous identification of suspected infectious cases is crucial to control the recent pandemic caused by the novel human coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). Real-time polymerase chain reaction (real-time PCR) technology cannot be implemented easily and in large scale in some communities due to lack of resources and infrastructures. Here, we report a simple colorimetric strategy derived from linker-based single-component assembly of gold nanoparticle-core spherical nucleic acids (AuNP-core SNAs) for visual detection of PCR products of SARS-CoV-2 ribonucleic acid (RNA) template. A palindromic linker is designed based on SARS-CoV-2 specific E gene to program the identical colloidal SNAs into large assemblies along with a distinct red-to-purple color change. The linker acts as a probe of SARS-CoV-2 RNA in conventional PCR reaction. In the presence of the correct template the palindromic linker, which is complementary to a short region within the target amplicon, is cleaved by 5'-exonuclease activity of deoxyribonucleic acid (DNA) polymerase. Cleavage of the palindromic linker during the amplification process inhibits the single-component assembly formation of SNAs. So, positive and negative viral samples produce simply red and purple colors in the post PCR colorimetric test, respectively. Evaluation of the samples obtained from cases with laboratory-confirmed SARS-CoV-2 infection revealed that our assay can rival with real-time PCR method in sensitivity.

Effect of Chemical Seed Treatment on Rice False Smut Control in Field.

Rice false smut, caused by the pathogen Ustilaginoidea virens, is a severe emerging disease in China. It affects not only the quality of rice but also yields of rice production. To make clear the effect of chemical seed treatment on the rice false smut control in fields, during 2014 to 2017, four fungicides with different modes of action were used to treat rice seeds contaminated by false smut balls. In rice-growing seasons, samples of rice tissues were taken for detection of U. virens by using a specific nested PCR method at different rice-growing stages. In addition, the occurrence of rice false smut was investigated at maturation stage. Results showed that U. virens in plant tissues decreased significantly at the seedling stage upon chemical seed treatment. Four chemical treatments decreased the detection rate significantly (P < 0.01) compared with the water treatment, but no significant difference was observed among four chemical treatments. However, the detection rate did not decease significantly at the tillering and booting stages. Similarly, the final occurrence of rice false smut did not show significant difference between each chemical and water treatment. These results suggested that chemical seed treatment had only limited efficacy in preventing occurrence of rice false smut; application of fungicides at the booting stage or integrated use of fungicides and agricultural practices might give a better control for this disease.

The Issue of Recurrently Positive Patients Who Recovered From COVID-19 According to the Current Discharge Criteria: Investigation of Patients from Multiple Medical Institutions in Wuhan, China.

The current discharge criteria for COVID-19 require that patients have 2 consecutive negative results for reverse transcription polymerase chain reaction (RT-PCR) detection. Here, we observed that recurrent positive RT-PCR test results in patients with 3 consecutive negative results (5.4%) were significantly decreased compared with those in patients with 2 consecutive negative results (20.6%); such patients reported positive RT-PCR test results within 1 to 12 days after meeting the discharge criteria. These results confirmed that many recovered patients could show a positive RT-PCR test result, and most of these patients could be identified by an additional RT-PCR test prior to discharge.

PCR-based screening, isolation, and partial characterization of motile lactobacilli from various animal feces.

BACKGROUND: Most lactobacilli found in animal intestines are generally non-motile, but there are few exceptions. Our previous work showed that Lactobacillus agilis BKN88, which is a highly motile strain originating from a chicken, takes advantage of motility in gut colonization in murine models, and thus motile lactobacilli likely have unique ecological characteristics conferred by motility. However, the ecology and habitat of gut-derived motile lactobacilli are still rarely understood. In addition, the limited availability of motile Lactobacillus isolates is one of the major obstacles for further studies. To gain insight into the ecology and habitat of the motile lactobacilli, we established a routinely applicable detection method for motile lactobacilli using PCR and subsequent selective isolation in semi-solid MRS medium for the collection of additional motile lactobacilli from animal feces. RESULTS: We applied the PCR detection using motile lactobacilli-specific primers, based on the motor switch protein gene (fliG) of flagella, to 120 animal feces, followed by selective isolation performed using 45 animal feces. As a result, motile lactobacilli were detected in 44 animal feces. In the selective isolation, 29 isolates of L. agilis and 2 isolates of L. ruminis were obtained from 8 animal species. CONCLUSIONS: These results indicated that motile lactobacilli are distributed in different animal species. Moreover, phylogenetic analysis of the L. agilis isolates suggests co-evolution with the host, and adaptation to a particular environmental niche.

Rapid and efficient detection methods of pathogenic swine enteric coronaviruses.

Porcine enteric coronaviruses (CoVs) cause highly contagious enteric diarrhea in suckling piglets. These COV infections are characterized by clinical signs of vomiting, watery diarrhea, dehydration, and high morbidity and mortality, resulting in significant economic losses and tremendous threats to the pig farming industry worldwide. Because the clinical manifestations of pigs infected by different CoVs are similar, it is difficult to differentiate between the specific pathogens. Effective high-throughput detection methods are powerful tools used in the prevention and control of diseases. The immune system of piglets is not well developed, so serological methods to detect antibodies against these viruses are not suitable for rapid and early detection. This paper reviews various PCR-based methods used for the rapid and efficient detection of these pathogenic CoVs in swine intestines. KEY POINTS: 1. Swine enteric coronaviruses (CoVs) emerged and reemerged in past years. 2. Enteric CoVs infect pigs at all ages with high mortality rate in suckling pigs. 3. Rapid and efficient detection methods are needed and critical for diagnosis.

Development of a Specific Polymerase Chain Reaction System for the Detection of Rice Orange Leaf Phytoplasma Detection.

Rice orange leaf disease (ROLD), caused by rice orange leaf phytoplasma (ROLP), is transmitted by leafhopper vectors Recilia dorsalis and Nephotettix cinticeps. ROLD severely devastates rice production in Asia. Accurate detection of the pathogen is important for disease management. Current nested polymerase chain reaction (nested PCR) method using phytoplasma universal primers is widely used to detect phytoplasmas; however, it has shortcoming of inconvenience and inaccuracy, for it needs two round of PCR reactions and could produce false positive results due to nontarget amplification. In this study, we developed a PCR assay using a set of primers designed based on the ROLP genome sequence to amplify house-keeping gene FtsH-1 in rice and leafhopper vector samples. This method is simple and rapid, and its sensitivity up to 10 pg/µl of total ROLP DNA. It also minimizes the false positive problem produced by nested PCR. This method was used to survey the geographic distribution of ROLD in southern China from 2016 to 2018. The results showed that the distribution areas and vector carrying rate of ROLD had gradually increased.

High-flux simultaneous screening of common foodborne pathogens and their virulent factors.

Rapid and sensitive detection techniques for foodborne pathogens are important to the food industry. However, traditional detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 4-7 days to complete. In this study, we described a high-flux polymerase chain reaction (PCR) method for simultaneous detection of nine targeted genes (rfbE, stx1, stx2, invA, oprI, tlh, trh, tdh, and hlyA) with multiplex strains. The designed primers were highly specific for their respective target gene fragments. As the selected primers follow the principles of similar melting and annealing temperature, all the targeted genes could be detected for one strain with the same PCR program. Combining with 96-well PCR plate, by adding a single different gene to each well in each row, both the ATCC strains (E. coli, Salmonella spp., V. parahaemolyticus, L. monocytogenes, P. aeruginosa, S. aureus) and the clinical strains (E. coli, P. aeruginosa, S. aureus) were simultaneously detected to carry their specific and virulence genes. Therefore, using 96-well PCR plate for PCR amplification might be applied to high-flux sequencing of specific and virulence genes.