Development and evaluation of a droplet digital PCR assay for the diagnosis of paucibacillary leprosy in skin biopsy specimens

Cheng, Xian; Sun, Lele; Zhao, Qing; Mi, Zihao; Yu, Gongqi; Wang, Zhenzhen; Sun, Yonghu; Wang, Chuan; Man, Chunhua; Fu, Fanghui; Liu, Hong; Zhang, Furen

doi:10.1371/journal.pntd.0007284

Cited by 26 publications

(20 citation statements)

References 34 publications

(49 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Genomic DNA was extracted from chyme by using QIAamp Fast DNA Stool Mini Kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and the concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The ddPCR was conducted in a QX200 Droplet Digital PCR system (Bio-Rad) as described previously 80 . In short, the final volume of each assay mixture was 20 μL, which contained 10 μL of 2x ddPCR supermix,100 nM primers and 4 μL of extracted DNA.…”

Section: Methodsmentioning

confidence: 99%

Puerarin enhances intestinal function in piglets infected with porcine epidemic diarrhea virus

Zhang

et al. 2021

Sci Rep

View full text Add to dashboard Cite

Puerarin has been reported to be an excellent antioxidant, anti-inflammatory and antimicrobial agent, but the potential effect of puerarin on porcine epidemic diarrhea virus (PEDV) is unclear. This study aimed to determine whether puerarin could alleviate intestinal injury in piglets infected with PEDV. A PEDV (Yunnan province strain) infection model was applied to 7-day-old piglets at 104.5 TCID50 (50% tissue culture infectious dose). Piglets were orally administered with puerarin at the dosage of 0.5 mg/kg body weight from day 5 to day 9. On day 9 of the trial, piglets were inoculated orally with PEDV. Three days later, jugular vein blood and intestinal samples were collected. Results showed puerarin reduced morbidity of piglets infected with PEDV. In addition, puerarin reduced the activities of aspartate aminotransferase and alkaline phosphatase, the ratio of serum aspartate aminotransferase to serum alanine aminotransferase, the number of white blood cells and neutrophils, and the plasma concentrations of interleukin-6, interleukin-8 and tumor necrosis factor-α, as well as protein abundances of heat shock protein-70 in PEDV-infected piglets. Moreover, puerarin increased D-xylose concentration but decreased intestinal fatty acid-binding protein concentration and diamine oxidase activity in the plasma of piglets infected with PEDV. Puerarin increased the activities of total superoxide dismutase, glutathione peroxidase and catalase, while decreasing the activities of myeloperoxidase and concentration of hydrogen peroxide in both the intestine and plasma of PEDV-infected piglets. Puerarin decreased mRNA levels of glutathione S-transferase omega 2 but increased the levels of nuclear factor erythroid 2-related factor 2. Furthermore, puerarin increased the abundance of total eubacteria (16S rRNA), Enterococcus genus, Lactobacillus genus and Enterobacteriaceae family in the intestine, but reduced the abundance of Clostridium coccoides in the caecum. These data indicate puerarin improved intestinal function in piglets infected by PEDV and may be a promising supplement for the prevention of PEDV infection.

show abstract

Section: Methodsmentioning

confidence: 99%

Puerarin enhances intestinal function in piglets infected with porcine epidemic diarrhea virus

Zhang

et al. 2021

Sci Rep

View full text Add to dashboard Cite

show abstract

“…leprae is very difficult, and traditional laboratory techniques, such as the BI, are far from sensitive and specific. In the absence of a true gold standard, PCR methods, such as quantitative PCR (qPCR), multiplex PCR, and droplet digital PCR assays, have been developed to aid the diagnosis of leprosy worldwide [18–23].…”

Section: Discussionmentioning

confidence: 99%

Develop and Field Evolution of Single Tube Nested PCR, SYBRGreen PCR Methods, for the Diagnosis of Leprosy in Paraffin-embedded Formalin Fixed Tissues in Yunnan Province, a Hyper endemic Area of Leprosy in China

Chen

Xing

et al. 2019

PLoS Negl Trop Dis

View full text Add to dashboard Cite

BackgroundDetection and pathology analysis of Mycobacterium leprae using skin biopsy tissues are essential for leprosy diagnosis and monitoring response to treatment. Although formalin fixation of patient tissues may not be ideal for molecular studies, biopsy samples are the most accessible material from suspected cases. Therefore, clinical molecular laboratories must be able to utilize formalin-fixed, paraffin-embedded (FFPE) material.ObjectiveTo determine the best molecular method for diagnosing and monitoring leprosy in FFPE specimens, we developed a single-tube nested PCR (STNPCR) (131 bp) and SYBRGreen PCR (101 bp) assay using primers for the M. leprae-specific repetitive element (RLEP) gene and evaluated the results compared to those using previously established RLEP primers (372 bp).MethodsFFPE biopsy samples obtained from 145 leprosy patients (during or after multidrug therapy (MDT)) and patients with 29 other confounding dermatoses were examined by the bacteria index (BI) and by simple PCR, STNPCR, and SYBRGreen PCR using primers amplifying a 372-bp, 131-bp or 101-bp fragment of RLEP, respectively.ResultsIn leprosy patients receiving MDT, STNPCR showed a highest specificity of 100% and a positive predictive value (PPV) of 100%. For multibacillary (MB), paucibacillary (PB) and all leprosy patients, the highest sensitivities were 91.42%, 39.13%, and 67.92%, negative predictive values (NPVs) were 8.57%, 60.36%, and 32.07%, and the highest accuracies were 93.93%, 62.67%, and 74.81%, respectively, higher than the results of SYBRGreen PCR and simple PCR. For post-MDT leprosy patients, SYBRGreen PCR showed the highest sensitivity of 50.0%, highest specificity of 100%, a PPV of 100%, an NPV of 100% and the highest accuracy of 83.72% for MB patients, which were higher than those of STNPCR and simple PCR. STNPCR showed the highest sensitivity of 26.66% and 34.48%, highest specificity of 100% and 100%, a PPV of 100% and 100%, NPV of 72.50% and 60.21%, and highest accuracy of 75.00% and 67.24% for PB and leprosy patients, respectively, higher than those of SYBRGreen PCR and simple PCR.ConclusionsThese findings suggest that STNPCR or SYBRGreen PCR (131-bp and 101-bp fragment amplification, respectively) for RLEP using FFPE specimens performs better as a diagnostic test and for monitoring response to MDT than does simple PCR based on 372-bp fragment amplification. Additionally, STNPCR showed increased sensitivity for PB diagnosis using FFPE specimens, which can be transferred remotely or retrieved from previous leprosy patients.

show abstract

“…Then the PCR was performed on a Bulk PCR Thermal Cycler using the following conditions: Pre-denature for 1 cycle at 95°C for 10 min; denature for 45 cycles at 95°C for 15 s; anneal and extend for 45 cycles at 60°C for 1 min. Finally, the fluorescence signal in each plate was analyzed by a QX200 Droplet Reader and Quanta-Soft™ Version 1.7.4 [10,25]. Each reaction adopted negative control and was performed in duplicate.…”

Section: Droplet Digital Pcr Reactionmentioning

confidence: 99%

Development of a droplet digital PCR method for detection of Streptococcus agalactiae

Zeng

Chen

et al. 2020

BMC Microbiol

View full text Add to dashboard Cite

Background: Streptococcus agalactiae (GBS) is the causative pathogen of puerperal sepsis in pregnant women and pneumonia, sepsis and meningitis in infants. Infection of GBS is responsible for the increased morbidity in pregnant women and the elderly, and bring challenges to clinical diagnosis and treatment. However, culture-based approaches to detect S.agalactiae is time-consuming with limited sensitivity. Besides, real-time quantitative PCR demands expensive instruments with tedious steps. Thus, we aim to establish a new detection method for more accurate and rapid detection of S.agalactiae. Results: The ddPCR primer targeted the CpsE gene showed better amplified efficiency in the reaction. The limit of detection for GBS DNA with ddPCR was able to reach 5 pg/μL. Moreover, no positive amplified signals could be detected in the reactions which served 11 non-GBS strains DNA as templates. Furthermore, the coefficient of variation of this method was 4.5%, indicating excellent repeatability of ddPCR assay. Conclusions: In our study, ddPCR was performed as a rapid detection of S.agalactiae with high sensitivity and specificity. This technique can promote the accuracy of the diagnosis of GBS infection and provide a scientific basis for clinical treatment.

show abstract

Development and evaluation of a droplet digital PCR assay for the diagnosis of paucibacillary leprosy in skin biopsy specimens

Cited by 26 publications

References 34 publications

Puerarin enhances intestinal function in piglets infected with porcine epidemic diarrhea virus

Puerarin enhances intestinal function in piglets infected with porcine epidemic diarrhea virus

Develop and Field Evolution of Single Tube Nested PCR, SYBRGreen PCR Methods, for the Diagnosis of Leprosy in Paraffin-embedded Formalin Fixed Tissues in Yunnan Province, a Hyper endemic Area of Leprosy in China

Development of a droplet digital PCR method for detection of Streptococcus agalactiae

Contact Info

Product

Resources

About