A Novel Semiquantitative Fluorescence-Based Multiplex Polymerase Chain Reaction Assay for Rapid Simultaneous Detection of Bacterial and Parasitic Pathogens from Blood
2005; Elsevier BV; Volume: 7; Issue: 2 Linguagem: Inglês
10.1016/s1525-1578(10)60554-5
ISSN1943-7811
AutoresAngamuthu Selvapandiyan, Katie Stabler, Nasim Akhtar Ansari, Stephen Kerby, Jenny Riemenschneider, Poonam Salotra, Robert Duncan, Hira L. Nakhasi,
Tópico(s)Yersinia bacterium, plague, ectoparasites research
ResumoA multiplex polymerase chain reaction assay was developed for the rapid simultaneous detection of category A select bacterial agents (Bacillus anthracis and Yersinia pestis) and parasitic pathogens (Leishmania species) in blood using the Cepheid Smart Cycler platform. B. anthracis (Sterne) and Yersinia. pseudotuberculosis were used in the assay for optimization for B. anthracis and Y. pestis, respectively. The specificity of the target amplicons [protective antigen gene of B. anthracis and rRNA genes of other pathogens or human (internal control)] was evaluated by staining the amplicons with SYBR Green I and determining their individual melting temperatures (Tm). As a novel approach for pathogen semiquantitation, the Tm peak height of the amplicon was correlated with a known standard curve of pathogen-spiked samples. This assay was able to detect DNA in blood spiked with less than 50 target cells/ml for all of the pathogens. The sensitivity of this assay in blood was 100% for the detection of Leishmania donovani from leishmaniasis patients and B. anthracis (Sterne) from symptomatic mice. The time necessary for performing this assay including sample preparation was less than 1.5 hours, making this a potentially useful method for rapidly diagnosing and monitoring the efficacy of drugs or vaccines in infected individuals. A multiplex polymerase chain reaction assay was developed for the rapid simultaneous detection of category A select bacterial agents (Bacillus anthracis and Yersinia pestis) and parasitic pathogens (Leishmania species) in blood using the Cepheid Smart Cycler platform. B. anthracis (Sterne) and Yersinia. pseudotuberculosis were used in the assay for optimization for B. anthracis and Y. pestis, respectively. The specificity of the target amplicons [protective antigen gene of B. anthracis and rRNA genes of other pathogens or human (internal control)] was evaluated by staining the amplicons with SYBR Green I and determining their individual melting temperatures (Tm). As a novel approach for pathogen semiquantitation, the Tm peak height of the amplicon was correlated with a known standard curve of pathogen-spiked samples. This assay was able to detect DNA in blood spiked with less than 50 target cells/ml for all of the pathogens. The sensitivity of this assay in blood was 100% for the detection of Leishmania donovani from leishmaniasis patients and B. anthracis (Sterne) from symptomatic mice. The time necessary for performing this assay including sample preparation was less than 1.5 hours, making this a potentially useful method for rapidly diagnosing and monitoring the efficacy of drugs or vaccines in infected individuals. Blood transfusion saves millions of lives each year. Eighty percent of the world's population lives in developing countries with an access to only 20% of the world's safe blood supply (World Health Organization, press releases, 2000, http://www.who.int/inf-pr-2000/en/state2000-09.html). Blood has the potential to be contaminated by numerous pathogens.1Chamberland ME Epstein J Dodd RY Persing D Will RG DeMaria Jr, A Emmanuel JC Pierce B Khabbaz R Blood safety.Emerg Infect Dis. 1998; 4: 410-411Crossref PubMed Scopus (11) Google Scholar Contamination of blood or blood products could be accidental, mainly due to donation from infected asymptomatic individuals or a deliberate bioterror attempt to spread the infectious agents. The organisms with maximum potential to be used as biothreats are usually highly virulent and often difficult to diagnose. Several bacterial and viral pathogens are listed by the Centers for Disease Control and Prevention as category A agents, which may appear in blood during infection. Some of them are gram-positive spore-forming bacteria, Bacillus anthracis and Clostridium botulinum that cause anthrax and botulism, respectively,2Kaplan EH Detecting bioterror attacks by screening blood donors: a best-case analysis.Emerg Infect Dis. 2003; 9: 909-914Crossref PubMed Scopus (9) Google Scholar gram-negative bacterium Yersinia pestis that causes plague,3Engelthaler DM Hinnebusch BJ Rittner CM Gage KL Quantitative competitive PCR as a technique for exploring flea-Yersina pestis dynamics.Am J Trop Med Hyg. 2000; 62: 552-560PubMed Google Scholar and viruses responsible for various hemorrhagic fevers.4Leroy EM Baize S Volchkov VE Fisher-Hoch SP Georges-Courbot MC Lansoud-Soukate J Capron M Debre P McCormick JB Georges AJ Human asymptomatic Ebola infection and strong inflammatory response.Lancet. 2000; 355: 2210-2215Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 5Oliveira De Paula S Malta Lima D Clotteau M Pires Neto Rd Rda J Lopes da Fonseca BA Improved detection of dengue-1 virus from IgM-positive serum samples using C6/36 cell cultures in association with RT-PCR.Intervirology. 2003; 46: 227-231Crossref PubMed Scopus (18) Google Scholar Death due to anthrax occurs when the bacteremia reaches 107 to 108 bacilli/ml of blood.6Swartz MN Recognition and management of anthrax—an update.N Engl J Med. 2001; 345: 1621-1626Crossref PubMed Scopus (294) Google Scholar Y. enterocolitica, a common contaminant of packed red blood cells, was responsible for 50% of all clinical sepsis episodes associated with the transfusion of red blood cells, of which 61% were fatal.7Klein HG Dodd RY Ness PM Fratantoni JA Nemo GJ Current status of microbial contamination of blood components: summary of a conference.Transfusion. 1997; 37: 95-101Crossref PubMed Scopus (78) Google Scholar Packed red blood cells or whole blood, usually stored at 4°C, can allow the growth of these gram-negative bacteria.8Wagner SJ Friedman LI Dodd RY Transfusion-associated bacterial sepsis.Clin Microbiol Rev. 1994; 7: 290-302Crossref PubMed Scopus (174) Google Scholar Among the bacteria mentioned above, B. anthracis and Y. pestis disseminate easily in the environment and result in high mortality rates.9Meehan PJ Rosenstein NE Gillen M Meyer RF Kiefer MJ Deitchman S Besser RE Ehrenberg RL Edwards KM Martinez KF Responding to detection of aerosolized Bacillus anthracis by autonomous detection systems in the workplace.MMWR Recomm Rep. 2004; 53: 1-12PubMed Google Scholar, 10Parkhill J Wren BW Thomson NR Titball RW Holden MT Prentice MB Sebaihia M James KD Churcher C Mungall KL Baker S Basham D Bentley SD Brooks K Cerdeno-Tarraga AM Chillingworth T Cronin A Davies RM Davis P Dougan G Feltwell T Hamlin N Holroyd S Jagels K Karlyshev AV Leather S Moule S Oyston PC Quail M Rutherford K Simmonds M Skelton J Stevens K Whitehead S Barrell BG Genome sequence of Yersinia pestis, the causative agent of plague.Nature. 2001; 413: 523-527Crossref PubMed Scopus (980) Google Scholar Concern about the usage of category A select organisms as bioweapons has increased because of recent intentional dissemination of B. anthracis spores in the United States.11Sobel J Khan AS Swerdlow DL Threat of a biological terrorist attack on the US food supply: the CDC perspective.Lancet. 2002; 359: 874-880Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar Although the average incubation period for such bacteria in the body before the onset of symptoms is only a few days (2 to 10 days),2Kaplan EH Detecting bioterror attacks by screening blood donors: a best-case analysis.Emerg Infect Dis. 2003; 9: 909-914Crossref PubMed Scopus (9) Google Scholar asymptomatic individuals could potentially donate infected blood in this window period. There are several protozoan parasites that are blood borne, each of which causes a unique severe disease and that are of concern to the blood supply. For example, parasites belonging to the trypanosomatid family such as Leishmania donovani and Leishmania major, cause the fatal visceral (kala-azar) and cutaneous leishmaniasis, respectively.12Handman E Leishmaniasis: current status of vaccine development.Clin Microbiol Rev. 2001; 14: 229-243Crossref PubMed Scopus (428) Google Scholar Similarly, Trypanosoma brucei and Trypanosoma cruzi are the causative agents for African sleeping sickness and Chagas disease, respectively.13Desquesnes M Davila AM Applications of PCR-based tools for detection and identification of animal trypanosomes: a review and perspectives.Vet Parasitol. 2002; 109: 213-231Crossref PubMed Scopus (135) Google Scholar Thousands of immigrants to the United States from Chagas disease endemic regions, carrying asymptomatic chronic T. cruzi infections, represent a reservoir population for potential transfusion transmission of T. cruzi.14Leiby DA Herron Jr, RM Read EJ Lenes BA Stumpf RJ Trypanosoma cruzi in Los Angeles and Miami blood donors: impact of evolving donor demographics on seroprevalence and implications for transfusion transmission.Transfusion. 2002; 42: 549-555Crossref PubMed Scopus (179) Google Scholar At least six T. cruzi transfusion-transmitted cases have been reported so far in the United States and Canada.14Leiby DA Herron Jr, RM Read EJ Lenes BA Stumpf RJ Trypanosoma cruzi in Los Angeles and Miami blood donors: impact of evolving donor demographics on seroprevalence and implications for transfusion transmission.Transfusion. 2002; 42: 549-555Crossref PubMed Scopus (179) Google Scholar Leishmaniasis currently threatens 1.5 to 2.0 million people annually with an estimated death toll of 50,000 persons/year in 88 countries around the world.12Handman E Leishmaniasis: current status of vaccine development.Clin Microbiol Rev. 2001; 14: 229-243Crossref PubMed Scopus (428) Google Scholar The intracellular parasite Leishmania is present in blood for an undefined period of time without showing any clinical symptoms during the initial phase of the disease.15Luz KG da Silva VO Gomes EM Machado FC Araujo MA Fonseca HE Freire TC d'Almeida JB Palatnik M Palatnik-de Sousa CB Prevalence of anti-Leishmania donovani antibody among Brazilian blood donors and multiply transfused hemodialysis patients.Am J Trop Med Hyg. 1997; 57: 168-171PubMed Google Scholar Such individuals can potentially donate infected blood, because Leishmania not only survives blood-banking storage conditions, but also retains its infectivity as demonstrated by studies in animal models, such as hamsters and dogs.16Palatnik-de-Sousa CB Paraguai-de-Souza E Gomes EM Soares-Machado FC Luz KG Borojevic R Transmission of visceral leishmaniasis by blood transfusion in hamsters.Braz J Med Biol Res. 1996; 29: 1311-1315PubMed Google Scholar, 17Giger U Oakley DA Owens SD Schantz P Leishmania donovani transmission by packed RBC transfusion to anemic dogs in the United States.Transfusion. 2002; 42: 381-383Crossref PubMed Scopus (45) Google Scholar Leishmania species such as L. donovani causing visceral disease have been shown to be transmitted by blood transfusion in several human cases.15Luz KG da Silva VO Gomes EM Machado FC Araujo MA Fonseca HE Freire TC d'Almeida JB Palatnik M Palatnik-de Sousa CB Prevalence of anti-Leishmania donovani antibody among Brazilian blood donors and multiply transfused hemodialysis patients.Am J Trop Med Hyg. 1997; 57: 168-171PubMed Google Scholar US army personnel stationed in endemic areas are exposed to Leishmania infection. In a recent Centers for Disease Control and Prevention/Department of Defense Morbidity and Mortality Weekly Report, ∼500 cases of cutaneous leishmaniasis and 2 visceral cases have been reported from US soldiers deployed in both Iraq and Afghanistan.18Aronson N Ananthakrishnan M Bernstein W Hochberg L Marovich M Ockenhouse C Yoon I Weina P Benson P Fischer J Hack D Hawkes C Polhemus M Wortmann G McEvoy P Neafie R Defraites R Herwaldt BL Update: Cutaneous leishmaniasis in U.S. military personnel—Southwest/Central Asia, 2002–2004.Morb Mort Wkly Rep. 2004; 53: 264-265PubMed Google Scholar Hence the Department of Defense and the American Association of Blood Banks implemented a 1-year deferral period for soldiers returning from Iraq after deployment and permanent deferral for a diagnosed case of leishmaniasis. This deferral does not extend to US soldiers in Afghanistan since they have been deferred because of Malaria. The Food and Drug Administration in the United States is in agreement with such a deferral policy as a measure to ensure the safety of the nation's blood supply from transmission of Leishmania (Blood Products Advisory Committee Meeting, December, 2003). Under the circumstances described above rapid and accurate detection of contamination or diagnosis of infection due to either bacterial or parasitic agents in blood is needed. Various methodologies have been reported to diagnose B. anthracis, Y. pestis, and Leishmania species with a wide range of specificity and sensitivity, mostly for an individual organism.3Engelthaler DM Hinnebusch BJ Rittner CM Gage KL Quantitative competitive PCR as a technique for exploring flea-Yersina pestis dynamics.Am J Trop Med Hyg. 2000; 62: 552-560PubMed Google Scholar, 13Desquesnes M Davila AM Applications of PCR-based tools for detection and identification of animal trypanosomes: a review and perspectives.Vet Parasitol. 2002; 109: 213-231Crossref PubMed Scopus (135) Google Scholar, 19Cheun HI Makino SI Watarai M Shirahata T Uchida I Takeshi K A simple and sensitive detection system for Bacillus anthracis in meat and tissue.J Appl Microbiol. 2001; 91: 421-426Crossref PubMed Scopus (31) Google Scholar, 20Fasanella A Losito S Trotta T Adone R Massa S Ciuchini F Chiocco D Detection of anthrax vaccine virulence factors by polymerase chain reaction.Vaccine. 2001; 19: 4214-4218Crossref PubMed Scopus (55) Google Scholar, 21Hoffmaster AR Meyer RF Bowen MD Marston CK Weyant RS Thurman K Messenger SL Minor EE Winchell JM Rassmussen MV Newton BR Parker JT Morrill WE McKinney N Barnett GA Sejvar JJ Jernigan JA Perkins BA Popovic T Evaluation and validation of a real-time polymerase chain reaction assay for rapid identification of Bacillus anthracis.Emerg Infect Dis. 2002; 8: 1178-1182Crossref PubMed Scopus (105) Google Scholar, 22Melo AC Almeida AM Leal NC Retrospective study of a plague outbreak by multiplex-PCR.Lett Appl Microbiol. 2003; 37: 361-364Crossref PubMed Scopus (14) Google Scholar, 23Neubauer H Meyer H Prior J Aleksic S Hensel A Splettstosser W A combination of different polymerase chain reaction (PCR) assays for the presumptive identification of Yersinia pestis.J Vet Med B Infect Dis Vet Public Health. 2000; 47: 573-580Crossref PubMed Scopus (35) Google Scholar, 24Ramisse V Patra G Garrigue H Guesdon JL Mock M Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA.FEMS Microbiol Lett. 1996; 145: 9-16Crossref PubMed Google Scholar, 25Rantakokko-Jalava K Viljanen MK Application of Bacillus anthracis PCR to simulated clinical samples.Clin Microbiol Infect. 2003; 9: 1051-1056Crossref PubMed Scopus (6) Google Scholar, 26Salotra P Sreenivas G Pogue GP Lee N Nakhasi HL Ramesh V Negi NS Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis.J Clin Microbiol. 2001; 39: 849-854Crossref PubMed Scopus (170) Google Scholar The present study describes the development and evaluation of a single-tube rapid multiplex polymerase chain reaction (PCR) for the simultaneous detection of B. anthracis, Y. pestis, and Trypanosomatida in blood. In the current study the sensitivity and specificity of a multiplex PCR assay for the detection of the three types of blood borne pathogens are assessed. Vaccine strain of B. anthracis (Sterne 34F2) (Colorado Serum Co., Denver, CO) was cultured in SG sporulation medium.27Caipo ML Duffy S Zhao L Schaffner DW Bacillus megaterium spore germination is influenced by inoculum size.J Appl Microbiol. 2002; 92: 879-884Crossref PubMed Scopus (46) Google Scholar Y. pseudotuberculosis (Pfeiffer) Smith and Thal [American Type Culture Collection (ATCC) no. 6905] and Y. enterocolitica strain 8081 were cultured in Luria-Bertani liquid medium.28Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor1989Google Scholar The bacterial cells were stored at −80°C according to standard procedure.28Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor1989Google Scholar To calculate cell concentration of the stored cultures, an aliquot of cultures was used to count cells by serial dilution and plating on Luria-Bertani agar plates. Different species of Leishmania and T. brucei from our laboratory stocks were cultured using published protocols29Joshi M Dwyer DM Nakhasi HL Cloning and characterization of differentially expressed genes from in vitro-grown 'amastigotes' of Leishmania donovani.Mol Biochem Parasitol. 1993; 58: 345-354Crossref PubMed Scopus (96) Google Scholar, 30Wang Z Morris JC Drew ME Englund PT Inhibition of Trypanosoma brucei gene expression by RNA interference using an integratable vector with opposing T7 promoters.J Biol Chem. 2000; 275: 40174-40179Crossref PubMed Scopus (450) Google Scholar and cells were counted using a Coulter particle counter (Beckman Coulter, Miami, FL). In vitro grown L. donovani axenic amastigote parasites (strain 1S, clone 2D, World Health Organization designation MHOM/SD/62/1S-CL2D)31Debrabant A Joshi MB Pimenta PF Dwyer DM Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics.Int J Parasitol. 2004; 34: 205-217Crossref PubMed Scopus (184) Google Scholar that closely resemble the amastigote stage of Leishmania found in human macrophages were used to spike Leishmania into blood samples.12Handman E Leishmaniasis: current status of vaccine development.Clin Microbiol Rev. 2001; 14: 229-243Crossref PubMed Scopus (428) Google Scholar The amplicons that were chosen are from the genes that have multiple copies in both selected pathogens and humans. The B. anthracis detection target is in the protective antigen gene located on plasmid pXO1, present also in the vaccine strain. The Y. pestis target is in the 16S rRNA gene region conserved in all of the Yersinia species. Similarly, to detect Leishmania species the target is in the conserved region of the 18S rRNA gene. To increase the confidence of the assay and to recognize the false-negatives that may arise because of a reaction failure, an internal control was used. The internal control target is in the human 18S rRNA gene. To establish a multiplex assay, we identified primer sets specific for these pathogen targets and the human ribosomal RNA control that have similar annealing temperatures and an optimal Mg2+ concentration using the Primer 3′ program.32van Baren MJ Heutink P The PCR suite.Bioinformatics. 2004; 20: 591-593Crossref PubMed Scopus (32) Google Scholar The length of all of the primers was between 18 to 23 bases with an optimum (50%) GC content. Optimum melting temperature (Tm) of the oligos was 58°C at a salt concentration of 50 mmol/L. The primers (sequences not provided for security reasons) of B. anthracis, Yersinia, and Leishmania were screened against the human RepBase library [a database of repetitive DNA sequence elements found in a variety of eukaryotic organisms ()] to avoid repetitive regions. The specificity of the primers and avoidance of cross-hybridization with human sequences was assured by testing in BLAST searches (). To avoid formation of primer homodimers and heterodimers, the primer sequences were tested in the Amplify 1.2′ program (). To discriminate the amplified products, care was taken while designing the oligos to select different sized amplicons that have different melting temperature values. The lengths of the four amplicons were 99 bp for B. anthracis, 121 bp for Yersinia, 163 bp Leishmania, and 141 bp for the internal control. The amplicon sequences were tested in the Mfold program to avoid secondary structures on DNA where the primers anneal.33Zuker M Mfold web server for nucleic acid folding and hybridization prediction.Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (10157) Google Scholar Total DNA from cultured B. anthracis (Sterne), Y. pseudotuberculosis, Leishmania species, and T. brucei was extracted, purified, and measured using the Genome DNA kit (BIO 101, Carlsbad, CA) and manufacturer's protocol. To determine the limit of detection, 200 μl of healthy anonymous donor human whole blood (National Institutes of Health blood bank) containing heparin as anti-coagulant was seeded with serial dilution of each pathogen and the DNA was extracted with the QIAamp DNA blood mini kit (Qiagen, Valencia, CA). To extract DNA from the blood of visceral leishmaniasis patients, 200 μl of blood was directly used for the extraction. However, to extract DNA from the mice infected with B. anthracis (Sterne) 10 μl of the blood was mixed with 190 μl of healthy human blood and used for extraction. The total processing time for a DNA extraction was less than 30 minutes. The DNA at the final step was dissolved in 50 μl of Qiagen elution buffer and 5 μl of this was used in each multiplex PCR reaction. A master mix was prepared (for each 25-μl reaction volume) containing 1× buffer (TaKaRa), 6 mmol/L MgCl2, 0.25 U Ex Taq (TaKaRa), 0.25 mmol/L dNTP, 0.3 μmol/L each primer, 1× SYBR Green I (vendor stock 10,000X; Molecular Probes, Eugene, OR), and water to 20 μl and aliquoted into Smart Cycler PCR reaction tubes (Cepheid, Sunnyvale, CA). Then 5 μl of template DNA was added to each tube. Cycling parameters were: preheat at 94°C for 150 seconds and then 45 two-step cycles of 94°C for 15 seconds and 64°C for 30 seconds. After the last amplification cycle, PCR products were analyzed by melting curve analysis in the Smart Cycler by slowly increasing the temperature to 95°C. Total reaction and melting curve analysis time for a sample was 55 minutes. The reactions were run in triplicate with appropriate controls. The fidelity of the amplified products of all of the organisms was confirmed by individually cloning the amplified products into pCRII-TOPO plasmid (Invitrogen, Carlsbad, CA) and sequencing. The lowest level of detection of each pathogen was determined by using the extracted DNA obtained from 200 μl of blood spiked with serial dilutions of cells from each individual pathogen (ie, 10,000, 1000, 100, 10, and 5 cells). To calculate the pathogen load in the blood, a standard curve based on the fluorescence peak height of the amplified products and pathogen cell number in blood was generated. The sensitivity of the multiplex assay was tested on the blood obtained from mice infected with B. anthracis (Sterne). The mice were inoculated intraperitoneally with a target dose (lethal dose 90%) of 2 to 2.5 × 107 spores of bacteria essentially as described previously.34Karginov VA Robinson TM Riemenschneider J Golding B Kennedy M Shiloach J Alibek K Treatment of anthrax infection with combination of ciprofloxacin and antibodies to protective antigen of Bacillus anthracis.FEMS Immunol Med Microbiol. 2004; 40: 71-74Crossref PubMed Scopus (45) Google Scholar Blood samples were collected in the presence of anti-coagulant from these infected mice at different time points after challenge. Samples from noninoculated mice were included in the assay as negative controls. Blood samples were collected from 11 kala azar (visceral leishmaniasis) patients from Bihar, India, and reporting to Safdarjung Hospital, New Delhi, India, at the pretreatment stage. The patients exhibited characteristic disease symptoms such as fever, hepatosplenomegaly, anemia, and leukopenia. Only patients that were clinically diagnosed for kala azar and found positive for the presence of the parasite in bone marrow aspirates at the pretreatment stage26Salotra P Sreenivas G Pogue GP Lee N Nakhasi HL Ramesh V Negi NS Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis.J Clin Microbiol. 2001; 39: 849-854Crossref PubMed Scopus (170) Google Scholar were used in this study. In addition, blood samples obtained from three patients after treatment with anti-leishmanial drugs were also included in the study. All of the collected blood samples were heparinized, their identity was blinded and stored at 4°C. The coding of the clinical status of the patient samples were broken after the assay was performed and results were then analyzed. All of the animal and human experiments were done under National Institutes of Health guidelines for animal and human protection (). Because the virulent strain of B. anthracis is a select agent, we used the animal vaccine (Sterne) strain for the assay standardization. Because both Y. pestis and Y. enterocolitica are infectious, the closely related species Y. pseudotuberculosis, which is not a human pathogen was used as a surrogate. L. donovani cells were used to test the primer set that has a target in all of the Leishmania and Trypanosoma species. To test the multiplex assay, the DNA of B. anthracis (Sterne) (100 fg), Y. pseudotuberculosis (100 fg), and L. donovani (50 pg) was mixed with human DNA [a 10th of the DNA (∼300 ng) obtained from 200 μl of blood] and used in a multiplex PCR. Amplification of all of the four amplicons was observed by the appearance of their expected fluorescence Tm peaks (Figure 1A). The Tm peak for B. anthracis (Sterne) was 82.2°C, for Y. pseudotuberculosis was 87.3°C, for L. donovani was 86.1°C, and for the human control was 88.8°C. The agarose gel picture of the PCR reaction shows the amplified products of all of the organisms with expected size DNA fragments (Figure 1B). The fidelity of the sequence of the amplified products was further confirmed by sequence analysis. After confirming the feasibility of the multiplex assay conducted using the DNA of the pathogens directly, the lower level of detection (LOD) of the pathogens in the blood was determined by doing the assay on DNA obtained from 200 μl of whole blood spiked with a known number of B. anthracis (Sterne) spores, 10,000, 1000, 100, 10, 5, and 0. A Tm peak corresponding to the amplified product of B. anthracis (Sterne) was observed at 82°C at all of the concentrations (Figure 2A). We also observed a gradual reduction in the height of the B. anthracis Tm peaks (Figure 2A) and the intensity of the amplified DNA bands of B. anthracis on the gel (Figure 2B) with the decreasing number of pathogen spores in the blood. However, in all of the reactions, the height of the internal control (human rRNA gene fragment amplification) Tm peak remained constant. We have used the height of the fluorescence peak above baseline of B. anthracis (Sterne) of different spore concentrations in plotting a standard graph for quantification (Figure 2C). All of the negative samples (nine) had baseline fluorescence. Similar studies on the determination of LOD were performed for the other two organisms, Y. pseudotuberculosis and L. donovani, and shown in Figure 2C. The analysis showed that the assay system can detect <10 cells per 200 μl of blood for all of the three pathogens. Each of the primer sets added in the multiplex reaction amplified its product only in the presence of the DNA from its corresponding organism. When the primer sets were tested individually on the templates of other organisms such as Escherichia coli and Bacillus cereus they did not amplify any DNA fragment (data not included). The primers neither cross-hybridized with each other nor did they react with any amplified products of other pathogens in the assay. They did not amplify any nonspecific fragments other than the four expected amplicons. However, Yersinia primers also amplified DNA from Y. enterocolitica (with Tm peak at 87°C) when tested in a separate reaction (data not shown). Similarly, the primers designed for Leishmania recognized, in addition to L. donovani, the DNA extracted from other closely related Leishmania and Trypanosoma species that cause different diseases, namely L. chagasi, L. infantum, and L. mexicana that cause visceral leishmaniasis; L. major and L. tropica that cause cutaneous leishmaniasis; L. braziliensis that causes mucocutaneous leishmaniasis; and Trypanosoma brucei that causes African sleeping sickness (data not shown). Multiplex PCR analysis was conducted with blood samples collected during a time course after infection from laboratory mice inoculated with B. anthracis (Sterne). The sample type and PCR results are shown in Table 1. Multiplex PCR conducted on the blood samples at 0 (prebleed) and 12 hours after the intraperitoneal injection of the spores of B. anthracis (Sterne) did not show the fluorescence peak for B. anthracis (Table 1). However, blood samples collected at 24-, 36-, and 48-hour time points after the inoculation were positive for the bacteria at an increasing rate with time after infection (Table 1). The bacteremia in the animal that tested PCR-positive after 24 hours of exposure was estimated to be 200 to 1000 cells/ml (cell concentration range obtained from the Tm peak standard curve) (Figure 2C). Similarly, four of five animals that were positive after 36 to 48 hours of exposure had bacteremia in their blood between 104 to 105 cells/ml. All these bacteremic mice were lethargic with scruffy hair and died within 24 hours.Table 1Multiplex Fluorescence PCR with Blood Samples from Mice Infected with the Spores of B. anthracis (Sterne)Blood collection time (hours after challenge)Total number of samples testedNumber of PCR-positive samples (% positives in parentheses)Approximate B. anthracis (Sterne) cell number in blood per ml060ND1260ND2441 (25)200 to 10003632 (66)1 × 104 to 1054822 (100)1 × 104 to 105ND, not detected. Open table in a new tab ND, not detected. The multiplex PCR assay was evaluated with the blood samples collected from
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