Multiplex PCR Assay For Detection Research Articles

Analytical and clinical validation of a multiplex PCR assay for detection of Neisseria gonorrhoeae and Chlamydia trachomatis including simultaneous LGV serotyping on an automated high-throughput PCR system.

For effective infection control measures for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), a reliable tool for screening and diagnosis is essential. Here, we aimed to establish and validate a multiplex PCR assay on an automated system using a dual-target approach for the detection of CT/NG and differentiation between lymphogranuloma venereum (LGV) and non-LGV from genital and extra-genital specimens. Published primer/probe sets (CT: pmpH, cryptic plasmid; NG: porA, opa) were modified for the cobas 5800/6800/8800. Standards quantified by digital PCR were used to determine linearity and lower limit of detection (LLoD; eSwab, urine). For clinical validation, prospective samples (n = 319) were compared with a CE-marked in vitro diagnostics (CE-IVD) assay. LLoDs ranged from 21.8 to 244 digital copies (dcp)/mL and 10.8 to 277 dcp/mL in swab and urine, respectively. A simple linear regression analysis yielded slopes ranging from -4.338 to -2.834 and Pearson correlation coefficients from 0.956 to 0.994. Inter- and intra-run variability was <0.5 and <1 cycle threshold (ct), respectively. No cross-reactivity was observed (n = 42). Clinical validation showed a sensitivity of 94.74% (95% confidence interval (CI): 87.23%-97.93%) and 95.51% (95% CI: 89.01%-98.24%), a specificity of 99.59% (95% CI: 97.71%-99.98%) and 99.57% (95% CI: 97.58%-99.98%), positive predictive values of 89.91% (estimated prevalence: 3.7%; 95% CI: 80.91%-95.6%) and 88.61% (estimated prevalence: 3.4%; 95% CI: 80.18%-94.34%), and negative predictive values of 99.81% (95% CI: 98.14%-100%) and 99.85% (95% CI: 98.14%-100%) for the detection of CT and NG, respectively. In conclusion, we established a dual-target, internally controlled PCR on an automated system for the detectiwon of CT/NG from genital and extra-genital specimens. Depending on local regulations, the assay can be used as a screening or a confirmatory/typing assay.IMPORTANCEChlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) represent a major global health burden, with the World Health Organization estimating that >128 million and >82 million people, respectively, were newly infected in 2020. For effective infection control measures, a reliable tool for sensitive diagnosis and screening of CT/NG is essential. We established a multiplex PCR assay for the detection of CT/NG and simultaneous discrimination between lymphogranuloma venereum (LGV) and non-LGV strains, which has been validated for genital and extra-genital specimens on a fully automated system. To increase assay sensitivity, a dual-target approach has been chosen for both pathogens. This strategy reduces false-positive results in oropharyngeal swabs due to the detection of commensal N. species that may harbor NG DNA fragments targeted in the PCR due to horizontal gene transmission following previous infection. In sum, the established assay provides a powerful tool for use as either a screening/diagnostic or a typing/confirmatory assay.

Optimization and evaluation of a multiplex PCR assay for detection of Staphylococcus aureus and its major virulence genes for assessing food safety.

Staphylococcus aureus is a natural commensal microflora of humans which causes opportunistic infections due to its large arsenal of exotoxins, invasion, immune evasion, and antibiotic resistance mechanisms. The primary goal of this study is to develop a multiplex PCR (mPCR) assay for simultaneous detection of Staphylococcus aureus (nuc) and its virulence genes coding for prominent exotoxins namely alpha hemolysin (hla), enterotoxins A (sea), enterotoxin B (seb), toxic shock syndrome toxin (tsst-1), and the gene coding for methicillin resistance (mecA). A competitive internal amplification control (IAC) was included in the assay to exclude the false negative outcomes. Highly specific primer pairs were designed for the target genes using in silico resources. At the outset, monoplex PCRs were standardized using reference S. aureus strains. Primer specificity to the target genes was authenticated through restriction digestion analysis of amplified PCR products. Multiplex PCR was optimized in increments of one gene starting with nuc and IAC amplified simultaneously using one pair of primers (nuc) in a competitive manner. The mPCR assay was found to be highly sensitive with a detection limit of ~10 CFUs per reaction for pure cultures. Multiplex PCR assay was further evaluated on the retail and processed food samples to test the prevalence of S. aureus and study their exotoxin profiles. Of the 57 samples examined, 13 samples (22.80%) were found to be contaminated with S. aureus whose DNA was extracted after a 6-h enrichment period. Among these, a high percentage of hemolytic and enterotoxin A positive strains were encountered. The mPCR assay developed in this study would be a useful tool for rapid and reliable monitoring of S. aureus for food quality testing and from clinical infections.

301. Use of a multiplex PCR assay for detection of respiratory co-infections in COVID-19 hospitalized non-ICU patients: an ace up a clinician’s sleeve

Abstract Background Bacterial co-infections in COVID-19 patients represent a significant challenge for clinicians and can impact outcomes. Rapid identification of bacterial co-pathogens improves management, and is crucial to avoid inappropriately administered antimicrobials. We evaluated the use of a multiplex PCR assay in the early detection of respiratory co-infections in COVID-19 hospitalized patients. Methods The study included non-ICU patients with high clinical suspicion of respiratory co-infection. Lower respiratory tract samples (sputum/bronchial secretions), were analyzed using BIOFIRE® FILMARRAY® pneumonia panel plus [(bioMerieux, USA), FA]. Specimens were considered as acceptable based on Gram stain. Conventional cultures were also performed. Results A total of 28 samples from 27 patients (20 males, median age 60 years, IQR 49-71) were analyzed. 8/27 patients were intubated, 5 were treated with high flow nasal canula oxygenation and 11 with high or low oxygen mask. 18 patients received dexamethasone. Co-infection was detected in 17/27 patients (62.9%). 11 specimens were collected in less than 48 hours from admission and no target was identified in 6/11. Detected pathogens and AMR genes, per sample tested, are presented in the table. Bacterial pathogens and any AMR genes were detected in 15 and 4 samples, respectively, leading to modifications of antimicrobial treatment. The semi-quantitative results along with patients’ clinical presentation assisted with differentiation of bacterial colonization versus infection, especially in cases where multiple targets were identified. De-escalation was implemented for 12 patients, for whom no co-infection or a viral co-pathogen (n=2) was detected. One patient was co-infected with another Coronavirus; further analysis, using the FA respiratory panel, detected Human coronavirus HKU-1 along with SARS-CoV-2. Median time until discharge or death was 13.5 days (IQR 9-24). Overall mortality was 33.3%. Detected pathogens, microbial load and AMR genes, per sample tested. Conclusion Implementation of FA assay proved effective for the rapid detection of respiratory co-infections in COVID-19 non-ICU patients. Molecular panel-based assays can contribute to timely adaptation of antimicrobial treatment, benefiting patient management and antibiotic stewardship strategies. Disclosures All Authors: No reported disclosures.

Molecular Detection of Bacterial Agents of Atypical Pneumonia among patients from six hospitals in Suez Canal region

Background: Reliable diagnosis of atypical pneumonia microbial agents is crucial since they do not respond to beta-lactams but to other groups of antibiotics. Therefore, empiric treatment with beta lactam groups will be ineffective for their eradication if no additional antibiotics as macrolides are administered to the management plan. This study aimed to determine prevalence of Mycoplasma pneumoniae (M. pneumoniae), Chlamydophila pneumoniae (C. pneumoniae) and Legionella pneumophila (L. pneumophila) among patients with atypical pneumonia, and drawing physicians' attention to the role of these pathogens as etiologic agents of atypical pneumonia in the Suez Canal region. Methods: Eighty-four atypical pneumonia cases were enrolled in this study. Respiratory samples were collected. Part of each specimen was inoculated onto blood, MacConkey, and chocolate agar plates, another part of specimens was processed for DNA extraction and multiplex PCR assay for detection of M. pneumoniae p1adhesion gene, C. pneumoniae outer membrane protein (ompA) gene, and L. pneumophila macrophage infectivity potentiator (mip) gene. Results: Out of the 84 atypical pneumonia cases, atypical bacteria were detected by multiplex conventional PCR in 12 (14%) cases and they all were L. pneumophila, 3 cases (4%) were mixed with Staphylococcus aureus, and 2 (2%) cases were mixed with Streptococcus pyogens. Mycoplasma pneumoniae and C. pneumoniae were not detected by PCR in our samples. Conclusions: Legionella pneumophila incidence is not low in our geographical region in patients with atypical pneumonia; so it is of pivotal importance to recruit sensitive and reliable molecular based techniques to detect and control this infection in healthcare environments.

Development of a Multiplex PCR Assay for Detection and Discrimination of Pathogenic and Saprophytic Leptospira in Water

Leptospirosis is a zoonosis prevalent in tropical countries and affects animals and humans alike. Leptospira interrogans, the causative organism for this waterborne infection, spreads through the urine of infected animals. There is a direct link between contaminated water and Leptospira outbreaks. This study reports a rapid assay to detect and differentiate pathogenic Leptospira from non-pathogenic in environmental water using multiplex PCR. The assay uses primers targeting the Lipl32 and Lipl21 gene. The multiplex PCR has been standardized using 11 pathogenic and one saprophytic serovar of Leptospira. The analytical sensitivity of the developed method was evaluated with different concentrations of template DNA. This method was used to screen water samples collected from 20 different sources from Chengalpattu town in Kancheepuram District, Tamil Nadu, India. Of the 20 water samples screened, 13 samples tested positive for pathogenic Leptospira, and seven samples tested negative. Four water samples were found to carry both pathogenic and saprophytic species. The developed multiplex PCR assay is highly useful for detecting and distinguishing pathogenic and saprophytic leptospires in water.

A real-time multiplex PCR assay for detection of the causative agents of rat bite fever, Streptobacillus moniliformis and zoonoticStreptobacillus species

Rat bite fever (RBF) caused by Streptobacillus moniliformis has been described as a diagnostic challenge. While it has a favorable prognosis with treatment, timely diagnosis is hindered by the lack of culture-free identification methods. Here we present a multiplex real-time PCR assay that detects the zoonotic Streptobacillus spp. as well as differentiate the primary causative agent of RBF, Streptobacillus moniliformis. The performance of this assay was evaluated using mock clinical specimens for blood, serum, and urine. Analytical sensitivity was determined to be 3-4 genome equivalents (GE)/µl for the zoonotic Streptobacillus spp. target, and 1-2 GE/µl for the S. moniliformis specific target. The assay correctly detected only the intended targets with no cross-reactivity identified. The pathogen was detected in all spiked matrices and not detected in the negative non-spiked specimens. This rapid diagnostic assay may permit quicker diagnosis of RBF patients.

Detection of clinically important non tuberculous mycobacteria (NTM) from pulmonary samples through one-step multiplex PCR assay

BackgroundThe burden of non-tuberculous mycobacterial (NTM) disease is increasing worldwide but still its diagnosis is delayed and it is mistaken as multidrug-resistant tuberculosis (MDR-TB).The present study was performed to develop a multiplex PCR assay for detection and identification of clinically most common NTM to the species level from pulmonary samples.ResultsOut of 50 isolates, 26 were identified as Mycobacterium kansasii (MK), 20 were identified as Mycobacterium abscessus (MA) and 4 were identified as Mycobacterium avium complex (MAC) through multiplex PCR and further confirmed by sequencing.ConclusionOur study showed that multiplex PCR assay is a simple, convenient, and reliable technique for detection and differential identification of major NTM species.

Evaluation of a multiplex PCR assay for detection of respiratory viruses and Mycoplasma pneumoniae in oropharyngeal swab samples from outpatients.

BackgroundRespiratory viruses, such as influenza viruses, initially infect the upper airways but can manifest as severe lower respiratory tract infections in high‐risk patients with significant morbidity and mortality. For syndromic diagnosis, several multiplex nucleic acid amplification tests have been developed for clinics, of which SureX 13 Respiratory Pathogen Multiplex Kit (ResP) can simultaneously detect 13 pathogens directly from airway secretion specimens. The organisms identified are influenza virus A, influenza virus A pdmH1N1 (2009), influenza virus A H3N2, influenza virus B, adenovirus, boca virus, rhinovirus, parainfluenza virus, coronavirus, respiratory syncytial virus, human metapneumovirus, Mycoplasma pneumoniae, and Chlamydia.MethodsThis study provides performance evaluation data of this assay by comparing with pathogen‐specific PCRs from oropharyngeal swab samples.ResultsTen pathogens were detected in this assay, of which rhinovirus, adenovirus, and influenza virus A pdmH1N1 (2009) were the most common. The overall agreement between the ResP and the comparator tests was 93.8%. The ResP demonstrated 86.5% agreement for positive results and 97.8% agreement for negative results.ConclusionThe ResP assay demonstrated a highly concordant performance comparing with pathogen‐specific PCRs for detection of respiratory pathogens in oropharyngeal swabs from outpatients and could aid in the diagnosis of respiratory infections in a variety of clinical scenarios.

Development of a multiplex PCR test-system for detection of BHV-1, BVDV, Chlamydia spp. and Mycoplasma spp.

This study describes development of a multiplex PCR assay for detection of BHV-1, BVDV, Chlamydia spp. and Mycoplasma spp. infections in bovines. The assay was developed using genomic DNA and RNA and four sets of PCR primers targeting 16S rRNA genes of Chlamydia spp., Mycoplasma spp., 5’-UTR of Bovine viral diarrhea virus, gE of Bovine herpesvirus-1, respectively. A total of 100 tissue samples were collected from cattle suspected to be infected with the viral and bacterial pathogens (BVDV, BHV-1, Chlamydia spp. and Mycoplasma spp.) from different regions of Ukraine. A part of sample was stored at –50°C for isolation of genomic DNA and RNA. The multiplex PCR assay was optimized in the study. The specific primers designed and used in the study were found sensitive and specific in amplifying target genes viz. 16S rRNA, gE, 5’-UTR of Chlamydia spp., Mycoplasma spp., BHV-1 and BVDV, respectively. The PCR primers used in the optimization of multiplex PCR assay for detection of Bovine viral diarrhea virus, Bovine herpesvirus-1, Chlamydia spp., Mycolasma spp. could amplify 221 bp, 111 bp, 386 bp, 279 bp products, respectively. Non specific amplification was not observed

Establishment and application of a multiplex PCR assay for detection of Rhizoctonia cerealis, Bipolaris sorokiniana, and Fusarium spp. in winter wheat

Rhizoctonia cerealis, Bipolaris sorokiniana, and Fusarium spp. are three wheat-damaging soil-borne fungal pathogens causing sharp eyespot, common root rot, and crown rot, respectively. This study aimed to develop a multiplex PCR (mPCR) system to simultaneously detect these fungal pathogens. The mPCR assay was performed on wheat seedlings collected from a field in the Shandong Province. Specific primers were designed on the basis of the sequences of following genes: internal transcribed spacer (ITS) of R. cerealis, ITS of B. sorokiniana, and translation elongation factor (TEF1-α) of Fusarium spp., generating amplicons of 174, 418, and 600 bp, respectively. The mPCR system was optimized through an orthogonal design L18 (37) (three levels of five factors: three sets of primers, Taq DNA polymerase, and dNTPs) and statistical analysis. No nonspecific products were obtained upon amplification of nontarget species of the fungi. Sensitivity analysis revealed that the limit of detection of the mPCR assay for each pathogen was 100 pg. The mPCR results were further confirmed via subsequent visual diagnosis. The system developed herein provides a rapid, effective, and valuable method to simultaneously detect three soil-borne pathogens, thus facilitating prompt treatment and prevention of root diseases of wheat.

Molecular detection of antibiotic resistance genes in multidrug-resistant Staphylococcus aureus isolates from dog dental plaque

Multidrug resistant Staphylococcus aureus strains are a major health care problem both in humans and animals. In this work we described three multiplex PCR assays for detection of clinically relevant antibiotic resistance genes in S. aureus isolated from dog dental plaques. Thirty dental plaque samples were collected; then cultural, biochemical and molecular tests performed for isolation and identification of S. aureus from samples. The antibiotic susceptibility of the isolates were checked by Kirby Bauer disc diffusion method and the prevalence of antibiotic resistance genes determined using multiplex PCR assay. As a result S. aureus was isolated from 18 dog plaque samples. Fifteen of these isolates were resistant to penicillin. The mecA gene was more prevalent than blaZ among penicillin-resistant bacteria. Ten of the isolates were resistant to tetracycline. The percentage of tetM was higher than tetK among them. Also, 10 of the isolates were resistant to cefazolin among them bla TEM detected in higher rate than blaSHV and blaOXA-1. Hence multiplex PCR assay is a suitable method for detection of antibiotic resistance patterns of S. aureus isolates.

Correction for "Evaluation of a multiplex PCR assay for detection of cytomegalovirus in stool samples from patients with ulcerative colitis" (World J Gastroenterol 2015; 21: 12667-12675).

[This corrects the article on p. 12667 in vol. 21, PMID: 26640344.].

Multiplex PCR assay for detection of Helicobacter pylori isolated from Iraqi dyspeptic patients

Helicobacter pylori (H. pylori) is one of the most common infections worldwide and is associated with gastric disorders. H. pylori is genetically unstable and this reflected on its virulence factors and type of diseases. Cytotoxin associated gene A (CagA) product is a major virulence factor is thought to be associated with gastric disease. It is injected into epithelial cells, undergoes phosphorylation by host cell kinases, and perturbs host signaling pathways. In the present study, we used multiplex PCR assay for rapid detection of H. pylori infection and for the determination of CagA genotypes directly from gastric biopsy specimens. Gastric biopsies were collected from 210 patients with dyspeptic symptoms. Genotypic analysis of H. pylori genes was found 91.17% for 16S rRNA, 87.25% for flagellin A, 89.21% for glmM (ureC) and cagA gene was detected in (39.21%) 40 biopsy specimens. Our study indicated that ureC (glmM) gene PCR is the most specific for the detection of H. pylori in gastric biopsy specimens compared with16S rRNA gene. There were significant differences (P ≤ 0.01) in cagA positive rate, among different diseases.

New colony multiplex PCR assays for the detection and discrimination of vancomycin-resistant enterococcal species

New colony multiplex PCR assays for detection of seven types of vancomycin-resistance determinants and eight types of Enterococcus species were developed. For 135 enterococcal isolates examined in this study, these assays showed high sensitivity and specificity, and could provide the rapid and accurate detection of vancomycin-resistant determinants and Enterococcus spp.

Development and application of a multiplex PCR assay for detection of the Clostridium perfringens enterotoxin-encoding genes cpe and becAB

Clostridium perfringens causes food-borne gastroenteritis following the consumption of contaminated food by producing C. perfringens enterotoxin (CPE) in the intestines. Recently, we reported a novel enterotoxin, binary enterotoxin of C. perfringens (BEC) in C. perfringens isolates, which caused two disease outbreaks in Japan. Consequently, in the event of food poisoning outbreaks caused by C. perfringens, it is now necessary to screen for both the cpe and becAB genes by diagnostic PCR. Here, we present a simple multiplex PCR method for simultaneous detection of cpe, becAB and a C. perfringens control locus, phospholipase C (plc). Applying this method, we investigated the prevalence of cpe- or becAB-carrying C. perfringens strains in human stool and bovine rectum swab samples. Using a total of 169 isolates, we found that the percentage of becAB-carrying strains was very small (0.59%), one-tenth that of cpe-carrying strains. The simple method presented in this study with high specificity and sensitivity to C. perfringens will be a useful tool to survey the global prevalence of becAB-carrying C. perfringens strains.

Development of RFLP-PCR and simple multiplex PCR assays for detection and differentiation of two species of hemotropic mycoplasmas in naturally infected dogs

Canine hemotropic mycoplasmas occur worldwide and are caused by the uncultivable hemotrophic bacteria; Mycoplasma haemocanis (Mhc) and Candidatus Mycoplasma haematoparvum (CMhp). In this study, the occurrence and differentiation of both causative agents in dogs that originated from southern Iran were investigated by employing restriction fragment length polymorphism (RFLP)-PCR and simple multiplex PCR methods based on RNase P RNA (rnpB) and 16S rDNA markers, respectively. The results showed that the prevalence of Mhc and CMhp among 100 examined samples were 6.0 and 4.0 %, respectively. Both PCR assays provide valuable molecular tools for the differentiation of CMhp and Mhc DNA in canine blood samples compared to 16S rDNA sequencing results. Also, sequence analysis of the genes (rnpB and 16S rDNA) and subsequent phylogenetic analysis were performed for both hemoplasmas in the Iranian dogs’ population. Comparison of the Iranian sequences with those for other isolates of canine hemoplasmas deposited in the GenBank database revealed a higher interspecific variability for rnpB marker (up to 31.22 %) compared to 16S rDNA sequence (up to 17 %).

Development of a multiplex PCR assay for detection of different bacterial pathogens associated with reproductive disorders in cattle and buffaloes

The current study was undertaken to develop a multiplex PCR assay for detection of Brucella, Leptospira, Listeria and Mycoplasma spp. associated with reproductive disorders in cattle and buffaloes. Four pairs of oligonucleotide primers chosen to amplify target DNA regions viz. 31kDa MEM protein in Brucella, 16S rRNA gene in Leptospira, hlyA gene in Listeria and 16S rDNA in Mycoplasma spp. produced amplicon sizes of 223-bp, 331 bp, 456 bp and 270 bp respectively. No amplification was observed when the multiplex PCR was tested against commonly prevalent and related species of bacteria. The sensitivity of the multiplex PCR assay was 116 pg for Brucella, Leptospira, Listeria and Mycoplasma spp. The developed assay was tested for direct detection of the agents in 30 clinical samples of uterine discharges and foetal stomach contents collected from aborted animals and animals with reproductive disorders. By multiplex PCR, out of 30 samples, nine samples of uterine discharges were positive only for Mycoplasma and 1 sample of foetal stomach content was positive for Brucella, Leptospira and Mycoplasma. The multiplex PCR described here appeared to be a rapid and ideal method for detection of all four organisms simultaneously in a single tube reaction and hence can be used for routine diagnostics.

Development of two real-time multiplex PCR assays for the detection and quantification of eight key bacterial pathogens in lower respiratory tract infections

The frequent lack of a positive and timely microbiological diagnosis in patients with lower respiratory tract infection (LRTI) is an important obstacle to antimicrobial stewardship. Patients are typically prescribed broad-spectrum empirical antibiotics while microbiology results are awaited, but, because these are often slow, negative, or inconclusive, de-escalation to narrow-spectrum agents rarely occurs in clinical practice. The aim of this study was to develop and evaluate two multiplex real-time PCR assays for the sensitive detection and accurate quantification of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. We found that all eight bacterial targets could be reliably quantified from sputum specimens down to a concentration of 100 CFUs/reaction (8333 CFUs/mL). Furthermore, all 249 positive control isolates were correctly detected with our assay, demonstrating effectiveness on both reference strains and local clinical isolates. The specificity was 98% on a panel of nearly 100 negative control isolates. Bacterial load was quantified accurately when three bacterial targets were present in mixtures of varying concentrations, mimicking likely clinical scenarios in LRTI. Concordance with culture was 100% for culture-positive sputum specimens, and 90% for bronchoalveolar lavage fluid specimens, and additional culture-negative bacterial infections were detected and quantified. In conclusion, a quantitative molecular test for eight key bacterial causes of LRTI has the potential to provide a more sensitive decision-making tool, closer to the time-point of patient admission than current standard methods. This should facilitate de-escalation from broad-spectrum to narrow-spectrum antibiotics, substantially improving patient management and supporting efforts to curtail inappropriate antibiotic use.

Multiplex PCR assay for detection of human somatotropin and interferon alpha2b genes in plant material

Using transgenic plants as factories for production of physiologically active human proteins arouses special concern because occasional escape of such transgenes into environment may cause health problems. Creation of plant varieties producing pharmaceutically valuable proteins should be accompanied by development of detection methods suitable for controlling the transgene behavior. Here we describe a multiplex PCR protocol for revealing of two human genes (encoding growth hormone and interferon alpha2b) that have been successfully introduced into plant genomes. The primer pair designed for detection of human growth hormone coding sequence amplifies fragments of different size from the full-length gene in the human genome and the intronless coding sequence usually used for plant transformation. Application of this primer pair may be recommended for ruling out false positive results due to sample contamination with human DNA. Such a control may be useful also in PCR analysis during establishing of transgenic plants carrying genes of human origin.

Detection of Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti, with two different multiplex PCR assays

We have developed 2 real-time multiplex PCR assays for detection of Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. The efficiency and sensitivity of each multiplex PCR assay was evaluated using field-collected Ixodes scapularis ticks that were positive for each of the pathogens, cloned plasmids harboring each of the PCR targets, and laboratory I. scapularis infected with B. burgdorferi B31. There was no difference in efficiency or sensitivity when comparing the multiplex PCR with the individual PCR reactions. If the 2 multiplex PCR assays are used in the same analysis, field-collected ticks that only harbor B. miyamotoi can also be identified. The multiplex assays are fast and cost-effective methods for screening and detecting pathogens in ticks, when compared to single-target PCR.