PCR-based Method For Detection Research Articles

Polyvalent detection of twelve viruses and four viroids affecting tomato by using a unique polyprobe

Non-radioactive molecular hybridization represents an attractive approach for the detection of multiple plant virus and/or viroids and a good alternative to the more extended serological and PCR-based detection methods. The use of polyprobes or riboprobes carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the detection of up to 10 different pathogens or the development of genus-specific probes. In the present article, the polyprobe technology has been adapted for the detection of the main viruses and viroids affecting tomato crops. To do this, three polyprobes have been developed covering four viroids (Poly4), twelve viruses (Poly12) or the four viroids plus the twelve viruses (poly16). The detection limit of the three polyprobes was comparable to the individual probes allowing the detection of up to 0,2 pg/μl of viral or viroidal RNA. A survey of 50 field samples revealed that all positive samples detected with the individual probes were also detected with the corresponding poly12 (98%) or poly16 (100%) probes. The analysis of tomato seeds revealed that both, single and polyprobes, were able to detect an infected seed in a pool of 250 healthy seeds. Finally, a ring-test analysis among six laboratories revealed a high reproducibility of the non-radioactive molecular hybridization procedure using the three polyprobes. The use of this technology in the routine analysis of tomato samples is discussed.

Rapid identification of Erwinia amylovora and Pseudomonas syringae species and characterization of E. amylovora streptomycin resistance using quantitative PCR assays.

Erwinia amylovora and Pseudomonas syringae are bacterial phytopathogens responsible for considerable yield losses in commercial pome fruit production. The pathogens, if left untreated, can compromise tree health and economically impact entire commercial fruit productions. Historically, the choice of effective control methods has been limited. The use of antibiotics was proposed as an effective control method. The identification of these pathogens and screening for the presence of antibiotic resistance is paramount in the adoption and implementation of disease control methods. Molecular tests have been developed and accepted for identification and characterization of these disease-causing organisms. We improved existing molecular tests by developing methods that are equal or superior in robustness for identifying E. amylovora or P. syringae while being faster to execute. In addition, the real-time PCR-based detection method for E. amylovora provided complementary information on the susceptibility or resistance to streptomycin of individual isolates. Finally, we describe a methodology and results that compare the aggressiveness of the different bacterial isolates on four apple cultivars. We show that bacterial isolates exhibit different behaviors when brought into contact with various apple varieties and that the hierarchical clustering of symptom severity indicates a population structure, suggesting a genetic basis for host cultivar specificity.

Prospective Implementation of a Point-of-Care PCR-Based Detection Method to Guide Antibiotic Use Prior to Prostate Biopsy Compared to Targeted Prophylaxis and Physician Choice

ObjectiveTo perform pilot testing regarding implementation of a point-of-care qPCR-based test (EST200) targeting bacterial clonal groups representing the majority of sepsis-causing Escherichia coli before prostate biopsy to determine antibiotic selection. Materials and MethodsAfter IRB approval, we obtained rectal swabs to compare real-time qPCR analysis on a Rotor-Gene Q instrument (Qiagen, Hilden, Germany) to standard culture on ciprofloxacin infused (10mg/L) MacConkey agar and susceptibility testing. Techniques are compared by an area under the receiver operative curve (AUC). ResultsA total of 140 men participated in the study, 102 prebiopsy cultures were utilized to guide prophylaxis. We did not meet our accrual for the randomized portion of the clinical study, yet we did randomized 38 men without prebiopsy cultures to physician choice of antibiotic versus PCR-based approach. Regarding predicting Fluoroquinolone Resistant (FQR) at biopsy, prebiopsy cultures had an AUC of 0.91 (95%CI 0.84-1.00, P > .001) and polymerase chain reaction (PCR) had an AUC of 0.71 (95%CI 0.58-0.84, P = .005) (AUC comparison; Z = 2.31, P = .02). PCR correctly identified 4 of 5 FQR specimens. The PCR test attained an AUC of 0.79 (95%CI 0.56-1.00, P = .044) for detection of total FQR on the day of the biopsy. Risk-based techniques may overcompensate with additional antibiotics (21% versus 0%, P = .10). ConclusionEST200 is a rapid PCR-based microbial detection system that has moderate ability to detect total FQR at the time of biopsy. Our study is underpowered, yet provide opportunities to improve the point of care PCR method, such as table tope testing in less than 20 minutes and include additional antibacterial resistant genes.

Comparison of methods for detection of Candida in bronchoalveolar lavage (BAL) of cancer patients

An accurate and rapid identification of Candida species in cancer patients with pulmonary symptoms can provide important information for effective treatment. Candida infections represent an increasing cause of morbidity and mortality in patients receiving immunosuppressive chemotherapy for cancer, organ transplantation or in immunocompromised. We used conventional methods such as culture, fermentation reactions, morphology and molecular methods based on the ribosomal DNA repetitive regions or the Internal Transcribed Spacer (ITS) for the identification of Candida species. Seventy bronchial specimens of Bronchoalveolar Lavage (BAL) from cancer patients at Shaukat Khanum Memorial Cancer Hospital and Research Center. Lahore, Pakistan were included in this study. Seventy cancer patients were diagnosed on the basis of histological profile. Candida detected by conventional methods using Sabouraud Dextrose Agar (SDA), potassium hydroxide (KOH) preparation, germ tube test, fermentation reactions and Gomori methanamine-silver stain (GMS). Thirty (42 %) positive isolates of Candida species were obtained by culture, twenty (28 %) isolates were germ tube test positive while thirty isolates (42 %) were positive by PCR method. In conclusion, the results of our study showed that the PCR based detection methods are significantly better and can detect Candida with more accuracy and specificity as compared to conventional methods. Our study would pave the path for optimization of protocols for detection of Candida in cancer patients.

Rapid PCR Assay for Detecting Common Genetic Variants Arising in Human Pluripotent Stem Cell Cultures.

Human pluripotent stem cells (hPSCs) are prone to acquiring genetic changes upon prolonged culture. Particularly common are copy number changes, including gains of chromosomes 1q, 12p, 17q, and 20q, and/or loss of chromosomes 10p and 18q. The variant cells harboring common genetic changes display altered behaviors compared to their diploid counterparts, thus potentially impacting upon the validity of experimental results and safety of hPSC-derived cellular therapies. Hence, a critical quality attribute in hPSC maintenance should include frequent monitoring for genetic changes arising in cultures. This in turn places large demands on the genotyping assays for detection of genetic changes. Traditional methods for screening cells entail specialized cytogenetic analyses, but their high costs and a lengthy turnaround time make them impractical for high-throughput analyses and routine laboratory use. Here, we detail a protocol for a rapid, accessible, and affordable PCR-based method for detection of frequently occurring copy number changes in hPSCs. © 2019 by John Wiley & Sons, Inc.

Engineering of a thermostable viral polymerase using metagenome-derived diversity for highly sensitive and specific RT-PCR.

Reverse transcription is an essential initial step in the analysis of RNA for most PCR-based amplification and detection methods. Despite advancements in these technologies, efficient conversion of RNAs that form stable secondary structures and double-stranded RNA targets remains challenging as retroviral-derived reverse transcriptases are often not sufficiently thermostable to catalyze synthesis at temperatures high enough to completely relax these structures. Here we describe the engineering and improvement of a thermostable viral family A polymerase with inherent reverse transcriptase activity for use in RT-PCR. Using the 3173 PyroPhage polymerase, previously identified from hot spring metagenomic sampling, and additional thermostable orthologs as a source of natural diversity, we used gene shuffling for library generation and screened for novel variants that retain high thermostability and display elevated reverse transcriptase activity. We then created a fusion enzyme between a high-performing variant polymerase and the 5′→3′ nuclease domain of Taq DNA polymerase that provided compatibility with probe-based detection chemistries and enabled highly sensitive detection of structured RNA targets. This technology enables a flexible single-enzyme RT-PCR system that has several advantages compared with standard heat-labile reverse transcription methods.

CDNA Display Mediated Immuno-PCR (cD-IPCR): A Novel PCR-based Antigen Detection Method.

Immuno-PCR (IPCR) is a powerful method in antigen detection where a PCR-amplifiable DNA reporter is conjugated to a specific antibody or an aptamer for the target molecule. In the development and application of IPCR, successful conjugation of a protein (an antibody) with a reporter DNA becomes challenging. To address this issue, we recently demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level. The cDNA display molecule for IPCR is generated first by transcribing the DNA that encodes the detection antibody into an mRNA by in vitro transcription. A puromycin DNA linker is then ligated to the mRNA and then in vitro translation and reverse-transcription are performed to generate the cDNA display molecule. The molecule is then directly used in antigen detection and subsequent qPCR. This method can be applied to detect various antigens in biological samples, if sequences of their single-domain antibodies (VHHs) or peptide aptamers are known.

Specificity and sensitivity of three PCR-based methods for detection of Erwinia amylovora in pure culture and plant material

Three PCR methods, referred in this study as ?conventional?, ?nested? and ?chromosomal? PCR and suggested for routine detection of Erwinia amylovora in pure culture and plant material, were evaluated according to their specificity and sensitivity. Specificity of PCR methods was analyzed by using 42 strains of E. amylovora, originating from different locations and plant species, with diverse PFGE profiles, representing distant populations of the pathogen. Sensitivity of PCR protocols in pure culture was studied by using nine different concentrations of E. amylovora in sterile ultrapure water as a template in PCR reactions. In order to study inhibitory effect of plant DNA and other inhibitors on sensitivity of the three PCR methods bacterial dilutions were mixed with plant macerate of pear, apple and quince prior to the PCR reaction. In specificity assays, tested PCR protocols were able to detect all E. amylovora strains regardless of the host of the strain, its origin or PFGE group, indicating primer specificity. On the other hand, sensitivity among tested methods varied, depending on bacterial concentration and selected plant material used in the PCR. When working with pure cultures nested PCR showed the greatest sensitivity by detecting 1.9 bacterial cells per PCR reaction, followed by detection limit of 9.5 cells per PCR reaction with conventional PCR and 1.9?105 cells/PCR reaction with chromosomal PCR. In spiked samples plant inhibitors either did not affect or they decreased the sensitivity of the PCR reaction, depending on the protocol and/or type of plant macerate. In our experiments, inhibitors from pear and quince macerates did not affect sensitivity of nested PCR, while apple macerate reduced its sensitivity by a factor of 10. Conventional PCR protocol was able to detect 95 cells/PCR reaction in pear and apple macerate, but only 9.5?103 cells/PCR in quince macerate. Greatest decrease in sensitivity of the PCR method was observed in spiked samples with chromosomal PCR since bacterial DNA was not detected in each of the spiked samples. Our research shows that all three PCR protocols are specific for detection of E. amylovora, but nested PCR proved to be most sensitive when working with pure cultures and plant material.

Application of ultra‐rapid qPCR and DNA chips for viral RNA detection and confirmation

The rapid and accurate detection of the presence of microorganisms, such as viruses, has been an important issue in the fields of public health, as well as agriculture. A PCR-based detection method has been developed and applied in these fields to determine the presence of specific pathogens. Although the major advantage of real-time PCR is the monitoring of amplification and ability to quantify the template genes, the method described here should solve the problem of nonspecific product synthesis. We obtained viral RNA from infected samples by freezing and thawing; we rapidly synthesized cDNA from RNA, and then amplified the cDNA by rapid PCR in 10Min. Finally, the PCR products were hybridized and quickly confirmed to be the target analyte on a DNA chip. Our newly proposed methods overcome the drawbacks of PCR-based detection and provide three additional advantages, namely, rapidly obtaining large amounts of RNA from samples, quickly detecting infective or pathogenic genes, and speedily confirming the detected exogenous genes. This application might be useful for detecting viral RNA and for the diagnosis of RNA virus-mediated diseases.

Early weaning completely eliminates porcine cytomegalovirus from a newly established pig donor facility for xenotransplantation.

For clinical xenotransplantation, transplants must be free of porcine cytomegalovirus (PCMV). Piglets become infected primarily in the perinatal period by the mother sow. While individual donor animals can be protected from infection by isolation husbandry, success is not guaranteed and this strategy poses the risk of undetected infections and raises animal welfare questions. Here, we present the establishment of a completely PCMV-negative pig herd for breeding donor animals for xenotransplantation. Eleven pregnant DanAvl Basic hybrid sows were purchased from a designated pathogen-free (DPF), PCMV-positive colony and transferred to a new pig facility at the Centre for Innovative Medical Models (CiMM) 4weeks prior to farrowing. At the age of 24hours, piglets were early-weaned and transferred to a commercially available Rescue Deck system dedicated to motherless rearing of piglets. Sows were removed from the facility. The PCMV status of F1-generation animals was determined at regular intervals over a period of 14months by a sensitive real-time PCR-based detection method testing blood, nasal swabs and cultured peripheral blood mononuclear cells (PBMCs). F1 sows were used as recipients of genetically modified embryos to generate a xenotransplant donor herd. Offspring were tested for PCMV accordingly. All offspring have remained PCMV negative over the whole observation period of 14months. A completely PCMV-negative pig herd for xenotransplantation has thus been successfully established.

A comparative analysis of microscopy and PCR based detection methods for Babesia and Trypanosoma infecting bovines and assessment of risk factors

The present study was carried out to evaluate and compare the status of infection and assessment of risk factors in 353 blood samples (144 cattle and 209 buffaloes) of bovines by PCR assay along with microscopic examinations (ME). ME revealed prevalence of Babesia bigemina and Trypanosoma evansi to be 22.91% and 0.69%, respectively in cattle and 12.44% and 0.95%, respectively in buffaloes. Conversely, PCR assay was able to detect 40.97% and 3.47 % prevalence of B. bigemina and T. evansi in cattle and 23.92% and 6.69% in buffaloes, respectively. The result revealed that the PCR assay was 100% sensitive and 82.9% specific when compared with ME for babesiosis and 100% sensitive and 95.42% specific for trypanosomosis. Multivariate logistic regression models showed that risk of babesiosis was significantly higher in cattle (Odds ratio (OR) =2.207, P=0.001) compared to buffaloes. The risk for surra in male buffaloes increased by 6.37 times (OR= 6.375, P=0.013). Conversely, risk of babesiosis was significantly lower in male cattle than females (OR= 0.467, P=0.044).

Simultaneous detection and quantification of 19 diarrhea-related pathogens with a quantitative real-time PCR panel assay

Acute diarrheal diseases are causes of global public health concern, especially in developing countries. A variety of diarrhea-associated microbial species, including bacteria, viruses, and protozoa, have been recognized. Simplified methods for detecting a wide range of diarrheagenic enteric microbes can clarify the etiology and aid in the diagnosis of diarrheal diseases. Here, we report a quantitative real-time (q)PCR-based method for simultaneous detection of 24 targets from 19 microbes suspected of causing diarrhea in stool specimens. We first selected the 24 oligonucleotide primer sets and hydrolysis probes conjugated with the fluorescent reporter dyes FAM, NED, or ABY, along with an internal control, and the passive reference dye ROX to establish a single-plate panel assay. The 12-duplex qPCR panel showed high linearity, with R2 values of 0.981–1.0 and limits of detection ranging from 1 to 103 fg for bacterial DNA (1–200 cells), 10–102 copies for viral DNA/RNA, and 10 fg for parasitic DNA (equivalent to approximately 1 parasite) per reaction. The accuracy and robustness of the assay was demonstrated in experiments using clinical stool specimens. This platform is low cost and easily customizable, and can be applied to various types of qPCR instruments and experimental designs for surveillance of acute diarrhea.

Characterization of a novel self-incompatibility allele in Malus and S-genotyping of select crabapple cultivars

Abstract Apples (Malus ×domestica Borkh.) are one of the largest economically significant fruit crop worldwide. Due to the self-incompatibility of most Malus cultivars, the apple industry relies on insect pollinators to aid in cross-pollination to improve seed and fruit set. The introduction of crabapples as pollinizers has become a popular practice to promote cross-pollination in commercial apple orchards. Genetic compatibility with a desired apple cultivar is a critical feature of effective pollinizers, but the S-genotypes of many crabapple cultivars used for cross-pollination have not yet been reported. In this study, seven crabapple cultivars were genotyped, and the genomic DNA of one novel S-allele in ‘Mt. Blanc’ and three variants of previously discovered alleles in ‘Manchurian’, ‘Snowdrift’ and ‘Indian Summer’ were characterized. Genomic DNA sequences were submitted to National Center for Biotechnology Information (NCBI) GenBank, and PCR-based detection methods were developed. The methods and results of this study aim to enrich S-genotyping methodologies and inform pollinizer-cultivar compatibility in commercial orchards.

Colorimetric PCR-Based microRNA Detection Method Based on Small Organic Dye and Single Enzyme.

microRNAs (miRNAs) have been a class of promising disease diagnostic biomarkers and therapeutic targets for their important biological functions. However, because of the high homology, interference from precursors (pri-miRNA, pre-miRNA), as well as limitations in the current assay technologies, it poses high demand and challenge for a specific, efficient, and economic miRNA assay method. Here, we propose a new miRNA detection method based on a label-free probe and a small organic dye with sequence dependence, realizing the sequence-specific and colorimetric detection of target miRNA. What is pleasantly surprising, only one enzyme is enough to propel the whole miRNA assay process, greatly simplifying the reaction component and detection process. Together with PCR amplification for the high enough sensitivity and three checks for specificity control, a detection limit of 5 fM was obtained and even one mutation could be discriminated visually. Overall, the new method makes much progress in convenience and economy of PCR-based miRNA assay method so that miRNA assay is going to be more friendly and affordable.

Equine infectious anemia virus in naturally infected horses from the Brazilian Pantanal.

Equine infectious anemia (EIA) has a worldwide distribution, and is widespread in Brazil. The Brazilian Pantanal presents with high prevalence comprising equine performance and indirectly the livestock industry, since the horses are used for cattle management. Although EIA is routinely diagnosed by the agar gel immunodiffusion test (AGID), this serological assay has some limitations, so PCR-based detection methods have the potential to overcome these limitations and act as complementary tests to those currently used. Considering the limited number of equine infectious anemia virus (EIAV) sequences which are available in public databases and the great genome variability, studies of EIAV detection and characterization molecular remain important. In this study we detected EIAV proviral DNA from 23 peripheral blood mononuclear cell (PBMCs) samples of naturally infected horses from Brazilian Pantanal using a semi-nested-PCR (sn-PCR). The serological profile of the animals was also evaluated by AGID and ELISA for gp90 and p26. Furthermore, the EIAV PCR amplified DNA was sequenced and phylogenetically analyzed. Here we describe the first EIAV sequences of the 5' LTR of the tat gene in naturally infected horses from Brazil, which presented with 91% similarity to EIAV reference sequences. The Brazilian EIAV sequences also presented variable nucleotide similarities among themselves, ranging from 93,5% to 100%. Phylogenetic analysis showed that Brazilian EIAV sequences grouped in a separate clade relative to other reference sequences. Thus this molecular detection and characterization may provide information about EIAV circulation in Brazilian territories and improve phylogenetic inferences.

Insertion of 275-bp SINE into first intron of PDIA4 gene is associated with litter size in Xiang pigs

The aim of the study was to investigate the SINE polymorphism in Xiang, Kele, Qianbei black, Jiangkouluobo, Large White, and Duroc pig breeds. The PCR based detection method was conducted to identify the short interspersed nuclear element (SINE) polymorphism in the PDIA4 gene. There were greater frequencies of the SINE−/− genotypes in Xiang pigs (55.9%) as compared with other pig breed groups. There was an association between this 275 bp SINE polymorphism and litter size (P = 0.003). The homozygous SINE+/+ genotype of the PDIA4 gene had a 1.45-piglets larger litter sizes compared to those with the homozygous SINE−/− genotype. Furthermore, there were assessments of mRNA and protein abundances as a result of PDIA4 gene expression in the ovaries of Xiang pigs for the three different SINE genotypes, and the results indicated that relative abundances of PDIA4 mRNA and protein was greater for the SINE−/− and SINE−/+ genotypes compared with the SINE+/+ genotype (P < 0.05). These findings suggested that the 275 bp SINE polymorphism might change the expression of the PDIA4 gene and could be used as a candidate DNA marker for the selection for litter size in Xiang pigs.

Real-Time PCR Assay for Detection of Kingella kingae in Children

Background Kingella kingae is a known cause of osteoarticular infections in children younger than 4 years of age, but it is not always recoverable in culture. Molecular methods are increasingly used for diagnosis. Methods To facilitate diagnosis of K. kingae septic arthritis, we developed a real-time polymerase chain reaction (PCR) assay for the detection of K. kingae that targets the repeat-in-toxin gene (rtxB). Results We present three pediatric patients with K. kingae septic arthritis at our institution who were diagnosed using the real-time PCR assay. All underwent arthrotomy with irrigation and debridement and were symptom-free after 3 weeks of therapy with β-lactam antibiotics. Cultures of synovial fluid or tissue grew K. kingae in two of three; K. kingae real-time PCR was positive in all three patients. In addition, 11 cases of K. kingae osteoarticular infection were diagnosed through Mayo Medical Laboratories using this assay. The limit of detection of the real-time PCR assay was 73.7 colony-forming unit (CFU)/µL for tissue and 1.3 CFU/µL for synovial fluid. Conclusions PCR-based detection methods are faster and more sensitive than conventional culture-based methods for the diagnosis of K. kingae osteoarticular infections in children.

Real-time PCR-based method for rapid detection of Aspergillus niger and Aspergillus welwitschiae isolated from coffee

Some species from Aspergillus section Nigri are morphologically very similar and altogether have been called A. niger aggregate. Although the species included in this group are morphologically very similar, they differ in their ability to produce mycotoxins and other metabolites and their taxonomical status has evolved continuously. Among them, A. niger and A. welwitschiae are ochratoxin A and fumonisin B2 producers and their detection and/or identification is of crucial importance for food safety. The aim of this study was the development of a real-time PCR-based method for simultaneous discrimination of A. niger and A. welwitschiae from other species of the A. niger aggregate isolated from coffee beans. One primer pair and a hybridization probe specific for detection of A. niger and A. welwitschiae strains were designed based on the BenA gene sequences, and used in a Real-time PCR assay for the rapid discrimination between both these species from all others of the A. niger aggregate. The Real-time PCR assay was shown to be 100% efficient in discriminating the 73 isolates of A. niger/A. welwitschiae from the other A. niger aggregate species analyzed as a negative control. This result testifies to the use of this technique as a good tool in the rapid detection of these important toxigenic species.

Highly Sensitive GMO Detection Using Real-Time PCR with a Large Amount of DNA Template: Single-Laboratory Validation.

Current genetically modified organism (GMO) detection methods allow for sensitive detection. However, a further increase in sensitivity will enable more efficient testing for large grain samples and reliable testing for processed foods. In this study, we investigated real-time PCR-based GMO detection methods using a large amount of DNA template. We selected target sequences that are commonly introduced into many kinds of GM crops, i.e., 35S promoter and nopaline synthase (NOS) terminator. This makes the newly developed method applicable to a wide range of GMOs, including some unauthorized ones. The estimated LOD of the new method was 0.005% of GM maize events; to the best of our knowledge, this method is the most sensitive among the GM maize detection methods for which the LOD was evaluated in terms of GMO content. A 10-fold increase in the DNA amount as compared with the amount used under common testing conditions gave an approximately 10-fold reduction in the LOD without PCR inhibition. Our method is applicable to various analytical samples, including processed foods. The use of other primers and fluorescence probes would permit highly sensitive detection of various recombinant DNA sequences besides the 35S promoter and NOS terminator.

Effects of Gamma Irradiation on the Molecular and Physical Detection Properties of Bacillus Spores

Gamma irradiation is a technique for inactivation of biological warfare agents (BWAs). Detection of these irradiated agents by sensor technologies may be affected,; altering sensitivity compared to that of non-irradiated organisms. Here for the first time, we aim to determine if spore morphology, or other physical or chemical properties beyond viability and PCR, is altered by the gamma irradiation process. This study analyzed common detection methods to determine the effects the irradiation process has on Bacillus thuringiensis subspecies kurstaki (Btk) spores compared to nonirradiated spores. Liquid concentrations of 108, 106, and 104 CFU ml-1 of Btk spores and one dry powder sample of 109 CFU g-1 were exposed to varying levels of gamma irradiation. Cell viability studies showed complete inactivation for all concentrations at doses of 5.43 kGy and higher. PCR analysis indicated no loss in sensitivity with increasing doses for both wet and dry spore samples. Visual inspection of the spores through scanning electron microscopy showed a change in morphology as the dosage of irradiation increased. With The inability to distinguish whole spores from cell debris occurred in at the 10.86 kGy samples level. Similarly, fluorescence readings remained stable among all but one dose with only the 10.86 kGy sample showing an increase in fluorescence. For PCR based detection methods, no bias towards irradiated versus non-irradiated spore samples exist while optical detection technologies are likely affected by the physical changes to spore morphology and rupturing. Results from this study demonstrate that gamma irradiation of Bacillus spores causes damage to the organism which may make them unrecognizable alter how they to are perceived by detection technologies. When using irradiated analyte to evaluate performance of detection technologies, the results may be impacted by the irradiation process.