Heterodera filipjevi continues to be a major threat to wheat production worldwide.Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease.In the pres...Heterodera filipjevi continues to be a major threat to wheat production worldwide.Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease.In the present study,a TaqManminor groove binder(TaqMan-MGB)probe-based fluorescence quantitative real-time PCR(qPCR)was successfully developed and used for quantifying H.filipjevi from DNA extracts of soil.The primers and probe designed from the obtained RAPD-SCAR marker fragments of H.filipjevi showed high specificity to H.filipjevi using DNA from isolatesconfirmed species of 23 Heterodera spp.,1 Globodera spp.and 3 Pratylenchus spp.The qPCR assay is highly sensitive and provides improved H.filipjevi detection sensitivity of as low as 4^(-3) single second-stage juvenile(J2)DNAs,10^(-3) female DNAs,and 0.01μgμL^(-1) genomic DNAs.A standard curve relating to the threshold cycle and log values of nematode numbers was generated and validated from artificially infested soils and was used to quantify H.filipjevi in naturally infested field soils.There was a high correlation between the H.filipjevi numbers estimated from 32 naturally infested field soils by both conventional methods and the numbers quantified using the qPCR assay.qPCR potentially provides a useful platform for the efficient detection and quantification of H.filipjevi directly from field soils and to quantify this species directly from DNA extracts of field soils.展开更多
Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resi...Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resistance necessitates updating diagnostic techniques to ensure higher sensitivity and specificity, especially with advancements in science and medicine. This study aimed to evaluate the prevalence of UTIs and antibiotic resistance profiles through urine culture, as well as to identify Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp. in urine samples using a molecular approach with multiplex real-time PCR. From May 3 to July 25, 2023, at the Pietro Annigoni Biomolecular Research Center (CERBA) and Saint Camille Hospital of Ouagadougou (HOSCO), 209 urine samples collected from patients with suspected UTIs were analyzed using both urine culture and multiplex real-time PCR. Among the 209 patients, 52.15% were male and 47.85% female, with an average age of 46.87 ± 21.33 years. Urine cultures revealed an overall UTI prevalence of 23.44%, with a prevalence of 8.13% in men versus 15.31% in women (P = 0.023). The bacterial prevalence rates were as follows: Escherichia coli (12.92%), Klebsiella spp. (7.18%), Enterobacter cloacae (1.44%), Staphylococcus aureus (0.96%), and other bacteria. Klebsiella spp. demonstrated 100% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, while Escherichia coli showed 96.2% and 65.4% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, respectively. PCR analysis of the target bacteria revealed mono-infection prevalence rates of Klebsiella pneumoniae (10.39%), Klebsiella oxytoca (7.79%), and Acinetobacter spp. (7.79%), along with a co-infection prevalence rate of Klebsiella pneumoniae/Acinetobacter spp. (1.30%). This study demonstrated that PCR, with its high sensitivity and specificity, could effectively distinguish Klebsiella pneumoniae from Klebsiella oxytoca and detect Acinetobacter spp. in less than 24 hours—something urine culture alone could not achieve. The relative ease of automating urine PCR testing, combined with its diagnostic accuracy and rapid turnaround time, makes it a valuable addition to modern medical practice for the laboratory diagnosis of UTIs.展开更多
基金financially supported by the National Natural Science Foundation of China(31972247)the Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences(ASTIP-2016-IPP-04)the Special Fund for Agro-scientific Research in the Public Interest,China(201503114)。
文摘Heterodera filipjevi continues to be a major threat to wheat production worldwide.Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease.In the present study,a TaqManminor groove binder(TaqMan-MGB)probe-based fluorescence quantitative real-time PCR(qPCR)was successfully developed and used for quantifying H.filipjevi from DNA extracts of soil.The primers and probe designed from the obtained RAPD-SCAR marker fragments of H.filipjevi showed high specificity to H.filipjevi using DNA from isolatesconfirmed species of 23 Heterodera spp.,1 Globodera spp.and 3 Pratylenchus spp.The qPCR assay is highly sensitive and provides improved H.filipjevi detection sensitivity of as low as 4^(-3) single second-stage juvenile(J2)DNAs,10^(-3) female DNAs,and 0.01μgμL^(-1) genomic DNAs.A standard curve relating to the threshold cycle and log values of nematode numbers was generated and validated from artificially infested soils and was used to quantify H.filipjevi in naturally infested field soils.There was a high correlation between the H.filipjevi numbers estimated from 32 naturally infested field soils by both conventional methods and the numbers quantified using the qPCR assay.qPCR potentially provides a useful platform for the efficient detection and quantification of H.filipjevi directly from field soils and to quantify this species directly from DNA extracts of field soils.
文摘Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resistance necessitates updating diagnostic techniques to ensure higher sensitivity and specificity, especially with advancements in science and medicine. This study aimed to evaluate the prevalence of UTIs and antibiotic resistance profiles through urine culture, as well as to identify Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp. in urine samples using a molecular approach with multiplex real-time PCR. From May 3 to July 25, 2023, at the Pietro Annigoni Biomolecular Research Center (CERBA) and Saint Camille Hospital of Ouagadougou (HOSCO), 209 urine samples collected from patients with suspected UTIs were analyzed using both urine culture and multiplex real-time PCR. Among the 209 patients, 52.15% were male and 47.85% female, with an average age of 46.87 ± 21.33 years. Urine cultures revealed an overall UTI prevalence of 23.44%, with a prevalence of 8.13% in men versus 15.31% in women (P = 0.023). The bacterial prevalence rates were as follows: Escherichia coli (12.92%), Klebsiella spp. (7.18%), Enterobacter cloacae (1.44%), Staphylococcus aureus (0.96%), and other bacteria. Klebsiella spp. demonstrated 100% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, while Escherichia coli showed 96.2% and 65.4% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, respectively. PCR analysis of the target bacteria revealed mono-infection prevalence rates of Klebsiella pneumoniae (10.39%), Klebsiella oxytoca (7.79%), and Acinetobacter spp. (7.79%), along with a co-infection prevalence rate of Klebsiella pneumoniae/Acinetobacter spp. (1.30%). This study demonstrated that PCR, with its high sensitivity and specificity, could effectively distinguish Klebsiella pneumoniae from Klebsiella oxytoca and detect Acinetobacter spp. in less than 24 hours—something urine culture alone could not achieve. The relative ease of automating urine PCR testing, combined with its diagnostic accuracy and rapid turnaround time, makes it a valuable addition to modern medical practice for the laboratory diagnosis of UTIs.
