NOVEL PCR ASSAYS FOR THREE FUR MITES IN NATURALLY INFECTED MICE WITHOUT ANIMAL SACRIFICE

2020 ◽  
pp. 1-10
Author(s):  
Chih-Lin Chou ◽  
Ying-Chien Cheng ◽  
Ming-Hseng Wang ◽  
Cho-Hua Wan
Keyword(s):  
Laboratory Mouse ◽  
Housekeeping Gene ◽  
Test Tape ◽  
Traditional Methods ◽  
Animal Sacrifice ◽  
Specific Pcr ◽  
Pcr Assays ◽  
Intractable Problem ◽  
Infestation Status ◽  
Higher Sensitivity

Rodent fur mite infestation is a persistent and intractable problem in laboratory rodent colonies, due to insensitive diagnostics, unrepresentative samples for testing and improper sentinel system. Myocoptes musculinus (COP), Myobia musculi (MOB) and Radfordia affinis (RDA) have been reported in laboratory mouse colonies. To improve the sensitivity and efficiency of fur mite detection, COP-specific PCR and MOB/RDA-specific PCR assays were developed to detect and differentiate these fur mites, with the existence of a rodent housekeeping gene. The COP-specific PCR and the MOB/RDA-specific PCR could specifically detect COP and MOB/RDA at as low as 10 copies, respectively. In comparison of the specific PCRs with traditional methods (pluck test, tape test and pelt exam) for fur mite diagnosis, 31 rodents and 17 cage environment samples were evaluated. In screening for the infestation status of various fur mites on individual animals, the specific PCR assays showed distinctly higher sensitivity and accuracy (100% and 100%) than those of the traditional methods (sensitivity: 25–80%, accuracy: 83.9–96.8%). Interestingly, by using cage wipe environmental samples, the specific PCR assays exhibited 100% sensitivity and accuracy in the fur mite detection and differentiation. The COP-specific and MOB/RDA-specific PCR assays developed in this study could be reliable alternatives for routine pathogen monitoring of laboratory mice and environments of animal facilities without animal sacrifice. [Chou C-L, Cheng Y-C, Wang MH, Wan CH, Novel PCR assays for three fur mites in naturally infected mice without animal sacrifice, Taiwan Vet J XX(X):XX–XX, 2020.

Development of Novel PCR Assays for Improved Detection of Enterovirus D68

2021 ◽  
Author(s):  
Tatsuki Ikuse ◽  
Yuta Aizawa ◽  
Hayato Takihara ◽  
Shujiro Okuda ◽  
Kanako Watanabe ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Clinical Manifestations ◽  
Assay Sensitivity ◽  
Acute Flaccid Myelitis ◽  
Specific Pcr ◽  
Enterovirus D68 ◽  
Pcr Assays ◽  
Higher Sensitivity

Enterovirus D68 (EV-D68) causes a range of clinical manifestations, including asthma-like illness, severe respiratory disease, and acute flaccid myelitis. EV-D68 has caused worldwide outbreaks since 2014 and is now recognized as a re-emerging infection in many countries. EV-D68–specific PCR assays are widely used for the diagnosis of EV-D68 infection; however, assay sensitivity is a concern because of genetic changes in recently circulated EV-D68. To address this, we summarized EV-D68 sequences from previously reported world outbreaks from 2014 through 2020 on GenBank, and found several mutations at the primer and probe binding sites of the existing EV-D68–specific PCR assays. Subsequently, we designed two novel assays corresponding to the recently reported EV-D68 sequences: an EV-D68–specific real-time and semi-nested PCR. In an analysis of 22 EV-D68–confirmed cases during a recent EV-D68 outbreak in Japan, the new real-time PCR had higher sensitivity than the existing assay (100% vs. 45%, P < 0.01) and a lower median Ct value (27.8 vs. 32.8, P = 0.005). Sensitivity was higher for the new non-nested PCR (91%) than for the existing semi-nested PCR assay (50%, P < 0.01). The specificity of the new real-time PCR was 100% using samples from non-EV-D68–infected cases (n = 135). In conclusion, our novel assays had higher sensitivity than the existing assay and might lead to more accurate diagnosis of recently circulating EV-D68. To prepare for future EV-D68 outbreaks, EV-D68–specific assays must be continuously monitored and updated.

scholarly journals CRISPR Is an Optimal Target for the Design of Specific PCR Assays for Salmonella enterica Serotypes Typhi and Paratyphi A

2014 ◽  
Vol 8 (1) ◽  
pp. e2671 ◽  
Author(s):  
Laetitia Fabre ◽  
Simon Le Hello ◽  
Chrystelle Roux ◽  
Sylvie Issenhuth-Jeanjean ◽  
François-Xavier Weill
Keyword(s):  
Salmonella Enterica ◽  
Specific Pcr ◽  
Optimal Target ◽  
Pcr Assays

scholarly journals Improved Primers for the Specific Detection of Leifsonia xyli subsp. xyli in Sugarcane Using a Conventional PCR Assay

Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3251-3258
Author(s):  
Sheng-Ren Sun ◽  
Jun-Lü Chen ◽  
Yao-Yao Duan ◽  
Na Chu ◽  
Mei-Ting Huang ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Dna Amplification ◽  
High Yield ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Saccharum Spp ◽  
Sensitivity Tests ◽  
Field Samples ◽  
Pcr Assays ◽  
Higher Sensitivity

Ratoon stunting disease (RSD), one of the most important diseases of sugarcane, is caused by the bacterium Leifsonia xyli subsp. xyli (Lxx). Lxx infects sugarcane worldwide and RSD results in high yield losses and varietal degeneration. It is highly challenging to diagnose RSD based on visual symptomatology because this disease does not exhibit distinct external and internal symptoms. In this study, a novel Lxx-specific primer pair Lxx-F1/Lxx-R1 was designed to detect this pathogen using a conventional PCR assay. These primers were then compared with four published Lxx-specific primers and one universal Leifsonia generic primer pair LayF/LayR. Sugarcane leaf samples were collected from Saccharum spp. hybrids in commercial fields (315 samples) and from germplasm clones of five Saccharum species and Erianthus arundinaceus (216 samples). These samples were used for comparative field diagnosis with six conventional PCR assays. Sensitivity tests suggested that the PCR assay with primers Lxx-F1/Lxx-R1 had the same detection limit (1 pg of Lxx genomic DNA) as the primer pairs Cxx1/Cxx2 and CxxITSf#5/CxxITSr#5 and had 10-fold higher sensitivity than the primer pairs Pat1-F2/Pat1-R2, LayF/LayR, and C2F/C2R. Comparison of PCR assays revealed that natural Lxx-infection incidence (6.1%) in field sample evaluation identified by Lxx-F1/Lxx-R1 primers was higher than incidences (0.7 to 3.0%) determined by other primer pairs. Moreover, no nonspecific DNA amplification occurred within these field samples with Lxx-F1/Lxx-R1 primers, unlike with the primer pairs Cxx1/Cxx2 and LayF/LayR. Diverse Leifsonia strains were identified by PCR detection with LayF/LayR primers in the field samples, whereas whether these Leifsonia strains were pathogenic to sugarcane requires further research. Our investigations revealed that the PCR assay with the newly designed primers Lxx-F1/Lxx-R1 could be widely used for RSD diagnosis and Lxx-pathogen detection with satisfactory sensitivity and specificity.

Specific PCR and real-time PCR assays for detection and quantitation of ‘Candidatus Phytoplasma phoenicium’

2015 ◽  
Vol 29 (1) ◽  
pp. 63-70 ◽  
Author(s):  
Maan Jawhari ◽  
Peter Abrahamian ◽  
Ali Abdel Sater ◽  
Hana Sobh ◽  
Patil Tawidian ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Specific Pcr ◽  
Pcr Assays

MULTIPLEX POLYMERASE CHAIN REACTION ASSAY FOR THE DETECTION OF FIVE COMMON ENDOPARASITE INFESTATIONS IN LABORATORY RODENTS

2020 ◽  
pp. 1-10
Author(s):  
Chien-Hao Chen ◽  
Ming-Hseng Wang ◽  
Cho-Hua Wan
Keyword(s):  
Multiplex Pcr ◽  
Laboratory Animal ◽  
Early Stage ◽  
Housekeeping Gene ◽  
Diagnostic Methods ◽  
Rrna Genes ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Laboratory Rodents ◽  
Infestation Status

Rodent pinworms, Spironucleus muris and Tritrichomonas muris are the endoparasites that should be monitored and excluded from laboratory animal colonies. Nevertheless, traditional diagnostic methods may not efficiently detect and accurately demonstrate the endoparasite infestation status. In this study, we developed a multiplex PCR assay targeting the rRNA genes to simultaneously detect and differentiate five endoparasites, including Syphacia obvelata, Syphacia muris, Aspiculuris tetraptera, Spironucleus muris, and T. muris, as well as a housekeeping gene in feces. The multiplex PCR could identify an equivalent infection of pinworm, Spironucleus muris and T. muris, with a detection limit of as few as 10 copies. Furthermore, dual infections with up to 100-fold differences and triple infections with 10-fold differences in parasite loads can also be detected. In comparison of traditional methods with the multiplex PCR assay, 76 rodents from 11 research colonies and 3 pet shops and additional 27 fecal samples from laboratory rodents were screened for the infestation status of the five endoparasites. The multiplex PCR had higher sensitivity (97.2–100%) and accuracy (99–100%) than those of the traditional antemortem (sensitivity: 83–100%; accuracy: 94–100%) and postmortem methods (sensitivity: 75–100%; accuracy: 92.1–100%). In addition, an early stage of S. obvelata contamination in a SPF laboratory animal colony was also successfully detected by this multiplex PCR assay. This Pinworm/Spironucleus/Tritrichomonas/Actin Multiplex PCR assay should be a powerful tool to screen endoparasite infestations in laboratory colonies without animal sacrifice.

Assessment of Minimal Residual Disease (MRD) by Qualitative RT-PCR (Q-PCR) vs Real-Time Quantitative RT-PCR (RQ-PCR) in a Phase II Study of Acute Promyelocytic Leukemia (APL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3262-3262
Author(s):  
Robert E. Gallagher ◽  
Esther L. Schachter-Tokarz ◽  
Dan A. Jones ◽  
Elihu H. Estey
Keyword(s):  
Phase Ii ◽  
Copy Number ◽  
Residual Disease ◽  
Blot Hybridization ◽  
Housekeeping Gene ◽  
Detection Sensitivity ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Increased Risk ◽  
Pcr Assays

Abstract Assessment of MRD in APL by Q-PCR analysis for the presence or absence of PML-RARα mRNA after the completion of consolidation therapy in treatment regimens employing all-trans retinoic acid (ATRA) and chemotherapy (CT) has provided a reasonably accurate but not infallible prognostic test for disease recurrence (DR). This assessment has been made at detection sensitivity levels up to 1 in 104, which is considered to have optimal predictive value, since non-quantified lower levels of PML-RARα mRNA may not be relevant to clinical outcome. In the current study, we compared the results of Q-PCR (touchdown PCR for 40 cycles followed by post-PCR blot hybridization with a RARα chemiluminescent probe) to results from RQ-PCR in 15 patients on a Phase II clinical trial designed to eliminate CT (low-risk/LR patients, WBC <10K) or minimize CT (high-risk/HR patients, WBC >10K), using combined ATRA and arsenic trioxide therapy. All patients achieved complete remission (CR) and have been followed for a median of 1.2 years. DR occurred in 0/8 LR, 0/1 unclassified and 2/6 HR patients. At CR, all Q-PCR and RQ-PCR assays were positive. For RQ-PCR, the normalized quotient (NQ) value of PML-RARα, i.e., the PML-RARα copy number divided by the housekeeping gene glyceraldehyde-3′-phosphate dehydrogenase (GAPDH) copy number, ranged widely from 1.5 x 10−6 to 7.0 x 10−3. Post-CR follow-up data were: Patient Group Number Q-PCR+ RQ-PCR+ NQ Range LR/Unclassified 9 0/24 16/24 9 x 10−9 – 1 x 10−7 HR/no DR 4 0/23 8/23 2 x 10−9 – 3 x 10−7 HR/DR 2 1/7 5/7 1 x 10−8 – 5 x 10−5 For the 2 HR/DR patients, the last pre-DR results were, respectively: Q-PCR, positive and negative; RQ-PCR, NQ = 5 x 10−5 and NQ = 2.3 x 10−7. In overall comparisons to RQ-PCR results, all Q-PCR assays were positive for NQ >10−5, 3/4 were positive in the NQ 10−6 to 10−5 range, and all were negative for NQ <10−6. Additionally, dilution-reconstruction experiments confirmed the stochastic nature of PCR assays near the detection limit and indicated that RQ-PCR was about 10-fold more sensitive than Q-PCR. Although larger studies are needed, our results suggest that HR patients who are Q-PCR-negative but RQ-PCR-positive in the >10−7 – <10−5 range at a critical post-CR checkpoint may be at increased risk of DR and, more generally, they suggest that the results of RQ-PCR testing at a critical checkpoint can be used in combination with clinical risk assessment to stratify the intensity of subsequent MRD monitoring in individual patients.

Concomitant characterization of androgen receptor (AC) and immune checkpoints (ICs) in cell-free (cf) DNA and RNA from patients with metastatic castration resistant prostate cancer (mCRPC).

2019 ◽  
Vol 37 (7_suppl) ◽  
pp. 286-286
Author(s):  
Sumanta K. Pal ◽  
Christopher Szeto ◽  
Sandeep K. Reddy ◽  
Roberto Nussenzveig ◽  
Richard Y. Lam ◽  
...  
Keyword(s):  
Clinical Care ◽  
Strong Association ◽  
Housekeeping Gene ◽  
Immune Checkpoints ◽  
Castration Resistant Prostate Cancer ◽  
Specific Pcr ◽  
Dna And Rna ◽  
Castration Resistant ◽  
Allele Specific ◽  
Allele Specific Pcr

286 Background: Immunotherapy (IO) appears to benefit a subset of patients (pt) with mCPRC, with recent data showing disease control > 6 mo in 11% of patients (De Bono et al ASCO 2018). We interrogate cfDNA/cfRNA from pt with mCRPC to ascertain relationships between AR and IC markers, and offer preliminary correlation with response to IC inhibitors (ICIs). Methods: Pt in this series were on active therapy for mCRPC and received cfDNA/cfRNA assessment for 26 analytes using the CLIA-certified NantHealth LiquidGPS platform during the course of routine clinical care. For this assay, 10 mL of blood were separately collected in DNA and RNA stabilizing solutions, respectively. DNA was amplified via mutant allele-specific blocker or allele-specific PCR assays. Wild type (WT) DNA was PCR amplified in an area where mutations had not been detected, and allele fractions were determined by the ratio of mutant to WT alleles. RNA was reverse transcribed to cDNA using random hexamer primers and amplified via gene specific assays relative to a stably expressed housekeeping gene, beta actin. Results: 103 pt were tested, median age 74y (range, 51-95). Expression was elevated for AR in 41%, and for AR-V7 in 3% pt. cfRNA detected expression for PD-L1, CTLA4, LAG-3 and TIM-3 in 60%, 52%, 72%, and 81% pt respectively. There was no strong association between AR and PD-L1 expression (Rho = 0.03, P = 0.77), or coincidence of detectable levels (OR = 1.4, p = 0.43). AR expression weakly inversely correlated with expression of CTLA4, LAG-3 and TIM-3. In 2 pts responding to ICI therapy, markedly increased PD-L1 and CTLA4 expression was noted. Multiple additional pts are receiving ICIs and correlation between expression of AR/IC markers and response will be presented. Conclusions: Unlike within the tumor microenvironment,expression ofcirculating AR and IC markers are independent biomarkers offering potentially more specific insights into combination versus sequential strategies for AR-directed tx and IO. Our preliminary observation of elevated circulating IC makers in the context of ICI response in mCRPC will be assessed in a larger series.

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