|SHORT RESEARCH COMMUNICATION
|Year : 2022 | Volume
| Issue : 2 | Page : 178-181
A Multiplex PCR assay for a differential diagnostic of rickettsiosis, Lyme disease and scrub typhus
Gaspar Peniche-Lara1, Isamara Moo-Salazar1, Karla Dzul-Rosado2
1 Laboratorio de Enfermedades Infecciosas y Parasitarias 1. Unidad Interinstitucional de Investigación Clínica y Epidemiológica, Facultad de Medicina, Universidad Autónoma de Yucatán. Yucatán, Mexico
2 Laboratorio de Enfermedades Emergentes y Re-emergentes. Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”. Universidad Autonoma de Yucatan. Yucatán, Mexico
|Date of Submission||21-Jan-2021|
|Date of Acceptance||06-Jan-2022|
|Date of Web Publication||08-Sep-2022|
Unidad Interinstitucional de Investigación Clinica y Epidemiologica, Facultad de Medicina, Universidad Autonoma de Yucatan, Avenida Itzaes por 58, Centro, 97000, Merida, Yucatan
Source of Support: None, Conflict of Interest: None
Background & objectives: Coexistence of tick-borne diseases in some regions in Latin America makes the diagnosis difficult due to shared initial signs and symptoms. Rickettsiosis, Lyme disease and recently, scrub typhus are gaining more importance. The objective of this study is to develop a multiplex-PCR assay for a differential diagnosis of rickettsiosis, Lyme disease and scrub typhus.
Methods: By using bibliographic and bioinformatic analysis, we identify candidate regions to perform the multiplex- PCR assay for Rickettsia sp., Borrelia burgdorferi and Orientia tsutsugamushi as well as identify optimal melting temperature and sensibility analysis.
Results: We identified specific primer pairs for Rickettsia sp, Borrelia burgdorferi and Orientia tsutsugamushi with different PCR fragment length but a common melting temperature, 58°C.
Interpretation & conclusion: We successfully developed a Multiplex PCR assay for differential diagnosis of rickettsiosis, Lyme disease and scrub typhus that could be a rapid and easy option in clinical and epidemiological practice.
Keywords: multiplex-PCR; rickettsiosis; human babesiosis; Lyme disease; scrub typhus
|How to cite this article:|
Peniche-Lara G, Moo-Salazar I, Dzul-Rosado K. A Multiplex PCR assay for a differential diagnostic of rickettsiosis, Lyme disease and scrub typhus. J Vector Borne Dis 2022;59:178-81
|How to cite this URL:|
Peniche-Lara G, Moo-Salazar I, Dzul-Rosado K. A Multiplex PCR assay for a differential diagnostic of rickettsiosis, Lyme disease and scrub typhus. J Vector Borne Dis [serial online] 2022 [cited 2022 Sep 28];59:178-81. Available from: https://www.jvbd.org/text.asp?2022/59/2/178/337506
| Introduction|| |
Tick, flea, and mite borne diseases are worldwide health problems that coexist some countries,,. Important tick-borne diseases like rickettsiosis or Lyme disease are common in some regions in Latin America,,. Recently, human babesiosis has been identified in Brazil, Bolivia, Colombia, and Mexico,,,. Other mite borne-diseases like scrub typhus have been recently identified in South Americas specifically in Chile by clinical cases and seroepidemiological studies, assuming the possibe presence of Orientia tsutsugamushi in other regions in Latin America. In all these diseases, signs and symptoms like fever, headache, exanthema, rash, diarrhea, presence of eschar are common and this makes the clinical diagnosis quite difficult in the early stages of the infection.
Rickettsiosis, Lyme disease and scrub typhus infections coexist in some areas in Latin America and this makes it necessary to develop diagnostic techniques that can simultaneously identify and differentiate these diseases. Multiplex-Polymerase chain reaction (PCR-Multiplex) is an excellent option to develop a molecular test for simultaneous amplification in a single tube which will let us provide a rapid, easy and cheap diagnostic test in laboratories with basic molecular infrastructure that will lead to specific treatments and medical care. In this study we have developed a multiplex-PCR option for differential diagnosis of rickettsiosis, Lyme disease and scrub typhus with a potential use for differential diagnosis for human and veterinary diseases.
| Material & Methods|| |
Primer selection and design
Primer pairs for each pathogen (Rickettsia spp., Borrelia burgdorferi, and Orientia tsutsugamushi) started with a bibliographic search for the identification of genes commonly employed for individual diagnosis of each pathogen, as well as primer pairs to identify a candidate gene fragment or a candidate primer pair for each pathogen in this study.
Candidate primer sequences obtained were evaluated for a theoretical melting temperature as well as fragment length obtained by using Bioedit sequence alignment editor 7.2.5 and pDraw32 DNA analysis software 1.1.14. An important inclusion criterion for primer pair selection was that the PCR products length obtained for each primer pair for each pathogen must have at least 100bp length of difference between them. Specificity of each primer, in order to prevent cross reaction between pathogens in study or other target, was evaluated using BLAST and Primer- BLAST in order to check if primers are complementary to different microorganism from the study.
Individual PCR standardization
We used DNA samples for each microorganism Ricketsia rickettsii, Sheila Smith strain, Babesia microti, Orientia Tsutsugamushi and Borrelia Burgdorferi. DNA controls of Rickettsia rickettsii and Orientia tsutsugamushi were provided by Donald Bouyer from the Department of Pathology at the University of Texas, Medical Branch in Galveston, Texas, USA. Employed DNA control from Borrelia burgdorferi were from our laboratory. Each primer pair were evaluated separately. We perform PCR reaction with different Tm (53–58°C) to identify the melting temperature that let us visualize the best band intensity. PCR reactions were prepared following the instructions manual of Maxima Hot Start PCR Master Mix (2X) (Thermo Scientific, USA). Amplification conditions inlcuded an initial denaturalization step of 94°C for 3 min followed by 30 cycles for 40 sec of 94°C, 40 sec of 53–57°C and 1 min of 72°C. Finally, we added a final extension step of 5 min of 72°C. Primer pairs employed in this step are presented in [Table 1]. In all experiments, negative control was added using sterile water as a sample. PCR products were visualized by agarose gel electrophoresis 1.5% using TBE 1x as a running buffer. Voltage and running time were 80V for 1.5 h. Agarose gels were stained with bromide ethidium and visualized in UV light.
Multiplex PCR standardization
In this procedure we employed the Maxima Hot Start PCR Master Mix (2X) (Thermo Scientific, USA). Amplification conditions were an initial denaturalization step of 94°C for 3 min followed by 30 cycles for 40 sec of 94°C, 40 sec of 56°C and 1 min of 72°C. finally, we added a final extension step of 5 min of 72°C. PCR products were visualized by agarose gel electrophoresis using the same procedures of the individual analysis.
To identify the minimum DNA concentration detected (limit of detection) by the multiplex-PCR developed, sensibility analysis was accomplished by repeating the Multiplex PCR assay using minimal DNA concentrations of each DNA sample using 2ng, 5ng, 10ng, 20ng, 50ng, 100ng.
Ethical statement: Not applicable
| Results|| |
Individual PCR standardization
Primer sequence and PCR product length are listed in [Table 1]. We selected a 58°C melting temperature for an individual analysis because we obtained the best band intensity for all primer pairs [Figure 1].
|Figure 1: 1.5% Agarose gel electrophoresis of PCR products obtained (employing 58°C as a melting temperature) from Orientia tsutsugamushi, Rickettsia rickettsii and Borrelia burgdorferi by using different DNA concentrations of each pathogen. Lane 1, 14 and 20: 100bp molecular weight maker. Lane 3-6 Orientia tsutsugamushi DNA using 10ng, 20ng, 50ng and 100ng. Lane 9-12: Rickettsia rickettsii DNA using 10ng, 20ng, 50ng and 100ng. Lane 15-18: Borrelia burgdorferi DNA using 10ng, 20ng, 50ng and 100ng.|
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Multiplex PCR standardization
Best band intensity for Multiplex PCR assay was obtained using a 58°C melting temperature [Figure 1]. In the sensibility analysis, we successfully amplified PCR products starting from 10ng to 100ng of DNA concentration from each pathogen [Figure 1]. Finally, evaluation of multiplex assay included analysis of one tube with all samples and primers simultaneously. We successfully observed a different PCR fragment from each pathogen in only one sample [Figure 2].
|Figure 2: 1.5% agarose gel electrophoresis of multiplex PCR designed showing the PCR fragments from each pathogen simultaneously. R: Rickettsia rickettsii, B: Borelia burgdorferi and O: Orientia tsutsugamushi. PCR products lengths are showed next to band lanes.|
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| Discussion|| |
During the last years, tick, flea and mite borne diseases like rickettsiosis, Lyme disease and scrub typhus have increased in terms of their worldwide distribution and, in consequence, has led to more human health problems,,,. Two points are important to respond to this increment in human cases (a) the lack of knowledge of these diseases by the medical community involved and (b) availability of diagnostic test to identify and differentiate between this vector borne diseases. The employment of differential diagnosis based on conventional Polymerase chain reaction like PCR-multiplex provides an opportunity by reducing the wait time for specific and differential results and, in consequence facilitate specific treatment. Till date this technology is commonly used in basic molecular biology laboratories and is comparable to other modern techniques like quantitative PCR (qPCR) or real-time PCR (RT-PCR) which have been successfully employed in the recently reported diseases.
Our Multiplex-PCR assay could be an excellent option for a differential diagnostic assay for rickettsiosis, Lyme disease and scrub typhus in areas where at least two of these diseases are present and could be a rapid and low- cost tool in epidemiological studies of tick, flea or mite diseases caused by Rickettsia spp, Borrelia burgdorferi or Orientia tsutsugamushi including their co-existence.
Conflict of interest: None
| Acknowledgements|| |
We are thankful to Donald Bouyer from the Department of Pathology at the University of Texas, Medical Branch in Galveston, Texas, USA for providing Rickettsia rickettsii and Orientia tsutsugamushi genomic DNA.
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[Figure 1], [Figure 2]