International Journal of Current Research and Review
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IJCRR - Vol 10 Issue 15, August, 2018

Pages: 15-19

Date of Publication: 16-Aug-2018


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Development of Polymerase Chain Reaction Assay for Targeting Cytochrome C Maturation F (ccmF) Gene of Leptospira

Author: Potdar Gayatri A., Dhotre Dheeraj P., Pol Sae S., Bharadwaj Renu S.

Category: Life Sciences

Abstract:Leptospirosis is a bacterial zoonotic disease caused by spirochetes of the genus Leptospira. The direct method of diagnosis of Leptospirosis has been so far by culture isolation but isolation rate of the microorganism from clinical specimens is low. The additional conventional method is the detection of antibodies which is done by ELISA, that fails to identify the infecting serovar. MAT is used as the gold standard, but it can only confirm the disease at a later acute phase because anti-Leptospira antibodies usually become measurable only 5 to 7 days after onset of illness. Thus, serology does not contribute to early diagnosis of Leptospirosis. To overcome these limitations, we developed PCR assay targeting ccmF gene of Leptospires using in-house designed RGf and RGr primers, with a product size of 910bp. The protocols were standardized. PCR could detect the target bacterial gene without any ambiguity. This simple, cost-effective and rapid method can be applied to detect causative Leptospira interrogans serovars from the isolates.

Keywords: PCR, ccmF gene, Molecular detection

Full Text:

Introduction:

Leptospirosis is a disease that is caused by spirochetes, members of the genus Leptospira. It is well illustrated as the frequent zoonosis in the world. Leptospirosis is most common in the tropics and distributed worldwide (except in the Polar Regions) (1). Under reporting is a major concern in analyzing the actual incidence of leptospirosis in many Asian countries (2). In India, it is directly related to either monsoons or poor sanitary conditions, with multiple epidemics reported (3–6).

For quick epidemiological response and also for timely treatment paradigms, rapid and simple assays that can reliably detect and distinguish Leptospira are necessary. Combining clinical proficiency and alertness with confirmatory laboratory back up noticeably increases the recognition of patients with Leptospirosis. As the clinical features are imprecise, laboratory test play a key role in diagnosis of Leptospirosis (7).

Since isolation rate of the microorganism from clinical specimens is low, often due to the prior indiscriminate use of antibiotics, serological techniques remain the cornerstone of diagnosis. The analysis of acute- and convalescent-phase sera by ELISA and the microscopic agglutination test (MAT). MAT is used as the gold standard, but it and can only confirm the disease at a later acute phase because anti-Leptospira antibodies usually become measurable only 5 to 7 days after onset of illness.

Hence, serology does not contribute to early diagnosis of Leptospirosis. Early diagnosis is obligatory because antibiotic treatment is most valuable when it is initiated early in the course of the disease. It is imperative to note that, in distinction to many of the resembling diseases (e.g. dengue), leptospirosis can easily be treated with antibiotics. This is provided that the diagnosis is confirmed in the first week of disease onset, when treatment with antibiotics is most effectual. Thus to facilitate preliminary treatment at the most effective time point, the availability of an accurate diagnostic test that is needed in the early acute phase of the disease.

Alternative methods were developed to assess the presence of leptospires in clinical samples like immune peroxidase staining (8), Immuno fluorescence staining (9) and DNA hybridization (10). These were useful methods for the detection of Leptospira, provided that the micro-organisms should present in fairly large numbers. These were not satisfactory for routine diagnostic purposes, mainly because of their lack of sensitivity.

Rapid recognition of small numbers of leptospires may become realistic due to precise amplification of Leptospiral DNA (11,12). PCR is a sensitive, specific, and rapid technique which has been successfully applied to the detection of several microorganisms and viruses in a variety of clinical specimens, including sputum, serum, cerebrospinal fluid, urine, faeces, and various tissues. PCR can rapidly confirm the detection in the early phase of the disease and before antibody titres are at detectable levels.

          Several PCR assays have been developed for the detection of leptospires species, and that are unable to detect Leptospira at serovar level. It is epidemiologically important to identify the infecting Leptospira serovars, as they may be associated with certain animal host species. So the appropriate control measures can be instituted.

          The aim of this study was to identify the gene with potential to enhance Leptospira serovar discrimination by sequencing-based methods. We used bioinformatics tools to check the discriminatory power of existing genes. Previously deposited sequence data were evaluated by phylogenetic analyses

Materials and Methods:

Computational determination of discriminatory powers of housekeeping genes

           Thirteen housekeeping genes were selected in a present study by comparison between two Leptospira genomic sequences available in gene bank (http://www.ncbi.nlm.nih.gov) those of L. interrogans serovars Copenhageni strain Fiocruz L1-130 chromosome I (accession number AE016823.1) and L. interrogans serovar Lai strain 56601 chromosome I (accession number AE010300.1)

           Multiple sequence alignments (MSA) for these sequences were carried out using CLUSTALX Version 2.1 software (13). Multiple Sequence Alignments were  manually edited to remove ambiguities using DAMBE software (14). Polymorphic sites were counted and percent polymorphism for each gene was determined (Table: 1).

Primer Design:

As ccmF gene was found to have maximum percent polymorphism (i. e. 5.8%), and was selected for further study.

Primer 3 software  was used to design primers for the hyper variable region of ccmF gene (15). In order to ensure optimal primer pair, primer length was set 20 bp, Tm was set at 450C to 600C, and the difference between forward and reverse primer sets was minimized (within 20C). The target value for primer GC content was 50–60%. No secondary structures within primers were allowed. All above parameters were manually checked by Generunner (http://ftparmy.com/130936-gene-runner.html) software.

         To check the lack of sequence homology with other bacterial or eukaryotic DNA sequences, designed primer sequences were checked for with a primer BLAST (NCBI, Bethesda, MD)(http://blast.ncbi.nlm.nih.gov/Blast.cgi) search. In silico designed primer pairs were synthesized by commercial manufacturer (Sci Fi biological) and were named as RGf and RGr primers.

Utility of primers:

 DNA extraction from standard serovars:

Five Leptospira interrogans serovars were obtained from National Leptospirosis Reference Centre, Regional Medical Research Centre in Port Blair, Andaman and Nicobar islands (listed in table: 2).

The serovars were grown in Ellinghausen–McCullough–Johnson–Harris (EMJH) media (BD, Sparks, MD) at 300C for 6–8 days. Leptospiral DNA was isolated from standard cultures with a Qiagen Blood and Cell Culture DNA Mini kit (Qiagen, Valencia, CA).

PCR standardization:

          A total reaction volume of 25 µL consisted of 10 µL Qiagen Master Mix, 1.5 µL of forward primer (2 pmol /µL stock), 1.5 µL of reverse primer (2 pmol /µL stock), 10 µL nuclease-free water and 2 µL DNA template. For negative controls, nuclease-free water was used instead of the DNA template. Samples were run for 2 min at 940C, followed by 30 cycles of 30 s at 94 0C, 30 s at 550C and 30 s at 72 0C on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA) At the end of the 30 cycles was an extension period of 7 min at 720C. PCR products were visualized on a 2% agarose gel and documented with a Gene Flash gel documentation system (Syngene, Frederick, MD). Each sample was run at least two times for reproducibility (Fig 1).

          Gene amplicons were sequenced using ABI 3730xl and sequences were submitted to NCBI to get accession numbers (Table 3).

 Phylogenetic tree of L. interrogans serovars for ccmF gene

Multiple sequence alignment (MSA) for sequences of standard leptospira serovars was carried out using CLUSTALX Version 2.1 software. Multiple Sequence Alignment was manually edited to remove ambiguities using DAMBE software.

Polymorphic sites were counted and percent polymorphism for ccmF gene was determined. Phylogenetic tree was constructed by Neighbour Joining  method using MEGA5 software package (16).

 Specificity testing of  primers

To check cross-annealing of primers with other species, ccmF primers designed were tested against the isolates of Bacillus cereus, Escherichia coli, Candida tropicalis, Salmonella typhi, Staphylococcus aureus and Mycobacterium tuberculosis.

DNA was isolated from these cultures with a Qiagen Blood and Cell Culture DNA Mini kit, and were tested by ccmF gene PCR.

Results:

ccmF gene (encoding Cytochrome C biogenesis protein) had maximum percent polymorphism i.e 5.8% among other genes studied.  Thus it was selected for further studies and primers were designed and PCR was standardized.

BLAST output of primers showed high degree of specificity for Leptospira species.

The size of the amplification products corresponded to the expected fragment size i.e. 910 bp.

The genetic distance between the L. interrogans serovars in the phylogenetic tree is represented by a bar which represents 0.005 nucleotide substitutions per alignment position among the serovars. This demonstrates variations in the sequence homology of ccmF gene which reflects in genetic diversity. All the Leptospira interrogans serovars under study were segregated from each other (Fig: 2). 

Primer sets produced only amplification products for standard Leptospiral serovars and not the other test organisms.

Discussion

In the last two decades, several PCR assays for the detection of Leptospires have been illustrated. PCR should generate an amplicon with sequence variability appropriate for phylogenic assessment for molecular epidemiological purposes.

The techniques such as, Restriction fragment length polymorphism analysis (17,18), ribotyping (17), pulsed-field gel electrophoresis (19) DNA hybridization (20), variable-number tandem-repeat analysis, arbitrarily primed PCR (APPCR), low-stringency single specific primer PCR (LSSP-

PCR), PCR restriction endo- nuclease analysis (PCR-REA) (21) etc. are available for Leptospira serovar identification.

However, fragment length polymorphism, ribotyping and pulsed-field gel electrophoresis are laborious, and time-intensive and require significant amounts of genomic DNA, whereas methods such as AP-PCR and LSSP-PCR result in complex banding patterns, DNA-DNA hybridisation is laborious and requires the use of radio-labelled isotopes. Hence cannot be used for routine diagnostic purposes.

Here we described the use of bioinformatics to identify novel gene i.e ccmF gene which is having potential of discrimination between Leptospira serovars.

PCR tests developed by other researchers also provide fast testing platforms, but the primers used can only differentiate between Leptospira species and have some disadvantages.

Primers presented in this study provide the ability to detect and differentiate between Leptospira interrogans serovars, that will be especially useful in epidemiological studies as the source for disease outbreaks.

ccmF gene codes for cytochrome c-type biogenesis protein CcmF. Cytochrome C is found in respiratory or photosynthetic electron transport chain, contain covalently bound heme. Bacterial C type cytochromes are extra cytoplasmic proteins and can be soluble and membrane bound. Presently we know 12 bacterial genes which control cytochrome C maturation ccm (ABCDEFGHI) res ABC and four genes that control redox state of the cell.

ccmF gene is housekeeping gene,  present in pathogenic and non pathogenic Leptospira which codes for cytochrome c-type biogenesis protein ccmF. It has an average length of 650 amino acids. As this gene has a housekeeping function, hence it is less susceptible to some forms of lateral gene transfer (22) .

The species L. interrogans is one of the dominant pathogen majorly found in the most infections related to Leptospirosis when compared to other pathogenic species like Leptospira borgpetersenii, Leptospira santarosai, Leptospira noguchii, Leptospira weilii, Leptospira kirschneri and Leptospira alexanderi. Cerqueira et al, 2009 demonstrated that the L. interrogans species is associated with severe human Leptospirosis, while the other strains like L. santarosai have shown their association with pigs and cattle. Hence, we first analyzed the various serovars related to species L. interrogans (23).

The potential of this assay must be balanced by a limitation: As ccmF gene is housekeeping gene, so it is present in non pathogenic Leptospira also. Hence DNA sequencing will be needed to discriminate between pathogenic and non pathogenic Leptospira.

Conclusion

The ccmF encoding gene had higher nucleotide divergence that allowed identification and also the potential for the discrimination between closely related Leptospiral serovars. The ccmF gene PCR may also likely to detect other L. interrogans  serovars hence it may be valuable tool for early diagnosis of  Leptospires directly from biological samples with clinical suspicion of leptospirosis. This new PCR could be useful in epidemiological studies to identify the source for disease outbreaks.

Acknowledgements

 The authors gratefully acknowledge Dr.Yogesh Shouche of Microbial Culture Collection, National Centre for Cell Sciences, Pune, MS, India for performing sequencing of Leptospira serovars.

We thank the Director, Dr.P. Vijayachari of National Leptospirosis Reference Centre, Regional Medical Research Centre (ICMR), Port Blair, India for providing us the leptospiral reference serovars.

Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature of this article has been reviewed and discussed.

Conflict of interests

The authors declare that they have no conflict of interests.

References:

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A Study by Varsha M. Shindhe et al. entitled "A Study on Effect of Smokeless Tobacco on Pulmonary Function Tests in Class IV Workers of USM-KLE (Universiti Sains Malaysia-Karnataka Lingayat Education Society) International Medical Programme, Belagavi" is awarded Best article of Vol 12 issue 14, July 2020
A study by Amruta Choudhary et al. entitled "Family Planning Knowledge, Attitude and Practice Among Women of Reproductive Age from Rural Area of Central India" is awarded Best Article for special issue "Modern Therapeutics Applications"
A study by Raunak Das entitled "Study of Cardiovascular Dysfunctions in Interstitial Lung Diseas epatients by Correlating the Levels of Serum NT PRO BNP and Microalbuminuria (Biomarkers of Cardiovascular Dysfunction) with Echocardiographic, Bronchoscopic and HighResolution Computed Tomography Findings of These ILD Patients" is awarded Best Article of Vol 12 issue 13 
A Study by Kannamani Ramasamy et al. entitled "COVID-19 Situation at Chennai City – Forecasting for the Better Pandemic Management" is awarded best article for  Vol 12 issue 12
A Study by Muhammet Lutfi SELCUK and Fatma entitled "Distinction of Gray and White Matter for Some Histological Staining Methods in New Zealand Rabbit's Brain" is awarded best article for  Vol 12 issue 11
A Study by Anamul Haq et al. entitled "Etiology of Abnormal Uterine Bleeding in Adolescents – Emphasis Upon Polycystic Ovarian Syndrome" is awarded best article for  Vol 12 issue 10
A Study by entitled "Estimation of Reference Interval of Serum Progesterone During Three Trimesters of Normal Pregnancy in a Tertiary Care Hospital of Kolkata" is awarded best article for  Vol 12 issue 09
A Study by Ilona Gracie De Souza & Pavan Kumar G. entitled "Effect of Releasing Myofascial Chain in Patients with Patellofemoral Pain Syndrome - A Randomized Clinical Trial" is awarded best article for  Vol 12 issue 08
A Study by Virendra Atam et. al. entitled "Clinical Profile and Short - Term Mortality Predictors in Acute Stroke with Emphasis on Stress Hyperglycemia and THRIVE Score : An Observational Study" is awarded best article for  Vol 12 issue 07
A Study by K. Krupashree et. al. entitled "Protective Effects of Picrorhizakurroa Against Fumonisin B1 Induced Hepatotoxicity in Mice" is awarded best article for issue Vol 10 issue 20
A study by Mithun K.P. et al "Larvicidal Activity of Crude Solanum Nigrum Leaf and Berries Extract Against Dengue Vector-Aedesaegypti" is awarded Best Article for Vol 10 issue 14 of IJCRR
A study by Asha Menon "Women in Child Care and Early Education: Truly Nontraditional Work" is awarded Best Article for Vol 10 issue 13
A study by Deep J. M. "Prevalence of Molar-Incisor Hypomineralization in 7-13 Years Old Children of Biratnagar, Nepal: A Cross Sectional Study" is awarded Best Article for Vol 10 issue 11 of IJCRR
A review by Chitra et al to analyse relation between Obesity and Type 2 diabetes is awarded 'Best Article' for Vol 10 issue 10 by IJCRR. 
A study by Karanpreet et al "Pregnancy Induced Hypertension: A Study on Its Multisystem Involvement" is given Best Paper Award for Vol 10 issue 09

List of Awardees

A Study by Ese Anibor et al. "Evaluation of Temporomandibular Joint Disorders Among Delta State University Students in Abraka, Nigeria" from Vol 13 issue 16 received Emerging Researcher Award


A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" from Vol 13 issue 06 received Emerging Researcher Award


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International Journal of Current Research and Review (IJCRR) provides platform for researchers to publish and discuss their original research and review work. IJCRR can not be held responsible for views, opinions and written statements of researchers published in this journal

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