Investigation of Botulinum Neurotoxin Types from Clostridium botulinum Causing A Recent Outbreak in Vietnam

Clostridium botulinum is one of the causes of undiagnosed sudden deaths in humans due to the lethal botulinum neurotoxins (BoNTs). Foodborne botulism rarely occurs in developed countries because of being closely monitored, in opposite to developing countries including Vietnam. In the August 2020 food poisoning outbreak in Vietnam, presence of Clostridium botulinum and BoNTs was identified by culture and mouse bioassay, however, information regarding the possible toxin types was unclear. To examine the types of toxin, we designed primers for specific amplification of gene regions encoding the light chain (LC) domains for both BoNT/A and BoNT/B. After optimization, the expected PCR products were sent for sequencing. The results showed that the sequence of gene encoding BoNT/A LC was 99.2% identical to the CB-27 strain. The sequence of gene encoding BoNT/B LC was approximately 98.8% identical to reference strains. Additionally, we analyzed the sequences of the inferred proteins and identified a substitution that resulted in an early stop codon as previously found in a defective form of BoNT/B. Collectively, we provided the first evidence for C. botulinum strain possessing A(b) type in this studied outbreak. Further enzyme activity and neutralization assays are necessary to validate this preliminary toxin typing.

Antifungal activity, identification and biosynthetic potential analysis of fungi against Rhizoctonia cerealis

Purpose Wheat sheath blight mainly infected by Rhizoctonia cerealis is one of the soil-borne fungal diseases of wheat worldwide and prevalent in major wheat growing areas in China at present. This study aimed to evaluate the antifungal activity of 163 endophytic fungi on R. cerealis. Antifungal strains were identified and their biosynthetic potential was analysed. Methods The antifungal activity of the strains was evaluated via dual-culture antagonism assay. The antifungal strains were identified on the basis of morphological characteristics and internal transcribed spacer gene sequencing. The polyketide synthases (PKSs) and nonribosomal peptide synthetase (NRPS) genes in antifungal strains were detected via specific amplification of chromosomal DNA. Result Twelve out of 163 fungal strains, including seven strains with matrix competition and five strains with antibiosis, were obtained. The twelve antifungal strains belonged to four genera: Alternaria, Ascochyta, Botryosphaeria, and Talaromyces. The inhibition rate of the seven strains with matrix competition was greater than 50%, with that of Botryosphaeria dothidea S2-33 being the highest at 84.6%. The inhibition zone of Talaromyces assiutensis R-03 amongst the five strains with antibiosis was the widest at up to 7 mm. Among the twelve antifungal strains, the strain S2-16 contained all the genes tested, five B. dothidea strains contained PKS-II and NRPS genes, two Alternaria alternata strains only contained PKS-II gene and the remaining four strains did not contain any. Conclusion Results demonstrated twelve potential strains for the biocontrol of wheat sheath blight. In particular, T. assiutensis R-03 was determined as a promising agent. The active substances secreted by antifungal strains may be produced by other biosynthetic pathways.

Case Report: Molecular Detection and Characterization of Trypanosoma cruzi in Ocular Tissue from Donors with Chagas Disease

Corneal transplantation is the most frequent transplant worldwide. Tissue characteristics allow storage and transport, even between continents, increasing its accessibility around the world. Donor infection with Trypanosoma cruzi is not defined as a corneal discarding factor, although the transplant is not recommended preventively, as in any infectious diseases. Herein, by means of polymerase chain reaction (PCR) strategies, we analyzed parasite presence in ocular tissue from 10 deceased donors with Chagas diseases. Among them, positive findings were obtained in corneas, scleras, and eye muscle samples of three, two, and one donor, respectively. Moreover, among the six T. cruzi defined populations, TcV and TcVI parasites were found in some samples based on group-specific amplification strategies. Our findings point out the actual possibility of T. cruzi transmission due to corneal transplantation and makes donor’s serological status knowledge mandatory regardless of graft provenance. Failing that, we suggest a posttransplant follow-up of recipients from seropositive donors.

Rapid and sensitive detection of Salmonella species targeting the hilA gene using a loop-mediated isothermal amplification assay

Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.

Comparative and Systematic Omics Revealed Low Cd Accumulation of Potato StMTP9 in Yeast: Suggesting a New Mechanism for Heavy Metal Detoxification

The metal tolerance protein (MTP) family is a very old family with evolutionary conservation and less specific amplification. It seems to retain the original functions of the ancestral genes and plays an important role in maintaining metal homeostasis in plant cells. We identified the potato MTP family members for the first time, the specific and conservative StMPTs were discovered by using systematic and comparative omics. To be surprised, members of the StMTP family seem to have mutated before the evolution of dicotyledon and monocotyledon, and even the loss of the entire subfamily (subfamily G6, G7). Interestingly, StMTP9 represents the conserved structure of the entire subfamily involved in toxic metal regulation. However, the gene structure and transmembrane domain of StMTP8 have undergone specific evolution, showing that the transmembrane domain (Motif13) located at the NH2 terminal has been replaced by the signal peptide domain, so it was selected as the control gene of StMTP9. Through real-time fluorescence quantitative analysis of StMTPs under Cd and Zn stress, a co-expression network was constructed, and it was found that StMTP9 responded significantly to Cd stress, while StMTP8 did the opposite. What excites us is that by introducing StMTPs 8/9 into the ∆ycf1 yeast cadmium-sensitive mutant strain, the functional complementation experiment proved that StMTPs 8/9 can restore Cd tolerance. In particular, StMTP9 can greatly reduce the cadmium content in yeast cells, while StMTP8 cannot. These findings provide a reference for further research on the molecular mechanism of potato toxic metal accumulation.

Diagnóstico de ectima contagioso em pequenos ruminantes através da Reação em Cadeia da Polimerase em Tempo Real

The virus of contagious ecthyma (CEV), also known as orf virus (ORFV) is the etiological agent of contagious ecthyma (CE) in sheep and goat and belongs to the Parapoxvirus genus, family Poxviridae. In some cases, CE can be confused with vesicular diseases so there is need for differentiation especially because, according to the standards of the National Program for the Eradication of FMD (PNEFA), goats and sheep are not vaccinated against Foot and Mouth Disease (FMD), acting as sentinel animals. Although initial studies have demonstrated the usefulness of the polymerase chain reaction (PCR) as a diagnostic test, there are no studies involving its use on Brazilian field samples, which may be genetically distinct from previously studied samples, as described in a study of restriction sites analysis of Brazilian CE samples. This work was conducted with the goal of standardizing a PCR (qPCR) test using SYBR Green I dye for molecular diagnosis of EC in DNA extracted from lesions of affected animal or cell culture inoculated in field samples. The products were detected with qPCR dissociation curve analysis which showed a peak at 88 ºC indicating that positive samples have only one specific amplification product. All DNA samples tested (29 animals crusts and their cell cultures) were positive in the qPCR. The qPCR was able to detect the DNA of at least 10,000 times dilution corresponding to 0.056 ng of DNA. It is believed that with the additional qPCR validations reported in this study, it can be used for differential diagnosis in the health surveillance of PNEFA.

Generation and Characterization of Monoclonal Antibodies Against Tth DNA Polymerase and its Application to Hot-Start PCR

Background: As a heat-resistant polymerase, Thermus thermophilus (Tth) DNA polymerase can be widely used in Polymerase Chain Reaction (PCR). However, its non-specific amplification phenomenon is serious, which greatly limits development. Objective: In this study, we prepared Tth monoclonal antibodies against Tth DNA polymerase and researched their application in hot-start PCR. Methods: Tth was recombinantly expressed and purified, and used as an antigen to immunize BALB/c mice to obtain monoclonal antibodies. The qualified monoclonal antibody and Tth are incubated for a period of time at a certain temperature to obtain the hot-start Tth. We tested the polymerase activity and exonuclease activity blocking performance of hot-start Tth. Finally, the hot start Tth was applied to one-step RT-PCR. Results: Tth with a purity of >95% was obtained, and ten monoclonal antibodies were obtained by immunization. After incubation, there were three monoclonal antibodies identified that could inhibit the polymerase activity of Tth at low temperature. Furthermore, these three antibodies have successfully eliminated non-specific amplification in practical applications. Conclusion: Three monoclonal antibodies were successfully validated. Among them, monoclonal antibody 9 has the best overall effect. They have the function of inhibiting at low temperature and releasing at high temperature, which can be used as Tth polymerase inhibitors in the field of molecular diagnostics.

RNase H-dependent amplification improves the accuracy of rolling circle amplification combined with loop-mediated isothermal amplification (RCA-LAMP)

The hybrid method upon combining rolling circle amplification and loop-mediated isothermal amplification (RCA-LAMP) was developed to quantify very small amount of different type of RNAs, such as miRNAs. RCA-LAMP can help detect short sequences through padlock probe (PLP) circularization and exhibit powerful DNA amplification. However, one of the factors that determines the detection limit of RCA-LAMP is non-specific amplification. In this study, we improved the accuracy of RCA-LAMP through applying RNase H-dependent PCR (rhPCR) technology. In this method, the non-specific amplification was suppressed by using the rh primer, which is designed through blocking the modification at the 3′end to stop DNA polymerase reaction and replacing the 6th DNA molecule from the end with RNA using RNase H2 enzyme. Traditional RCA-LAMP amplified the non-specific amplicons from linear PLP without a targeting reaction, while RCA-LAMP with rh primer and RNase H2 suppressed the non-specific amplification. Conversely, we identified the risk posed upon conducting PLP cyclization reaction using Splint R ligase in the RNA-targeting step that occurred even in the RNA-negative condition, which is another factor determining the detection limit of RCA-LAMP. Therefore, this study contributes in improving the accuracy of RNA quantification using RCA-LAMP.