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Year : 2021  |  Volume : 65  |  Issue : 4  |  Page : 369-374  

Standardization of an in-house multiplex real-time polymerase chain reaction for the simultaneous detection of Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex Virus 1 and 2, and Treponema pallidum infection among pregnant women

1 Sri Sakthi Amma Institute of Biomedical Research, Sri Narayani Hospital and Research Centre, Sripuram, Vellore, Tamil Nadu, India
2 Environmental Molecular Microbiology Research Laboratory, Department of Biotechnology, Thiruvalluvar University, Serkadu, Vellore, Tamil Nadu, India

Date of Submission23-Dec-2020
Date of Decision04-Jun-2021
Date of Acceptance22-Aug-2021
Date of Web Publication29-Dec-2021

Correspondence Address:
Mageshbabu Ramamurthy
Sri Narayani Hospital and Research Centre, Sri Sakthi Amma Institute of Biomedical Research, Sripuram, Vellore - 632 055, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijph.IJPH_1271_20

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Background: An in-house multiplex real-time polymerase chain reaction (PCR) was developed in two cocktails for the identification of six Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and Treponema pallidum (syphilis) (TORCH-S) agents, which causes congenital infection among pregnant women. Objective: Standardization and validation of an in-house multiplex real-time PCR assay for the detection of TORCH-S infection. Methods: This study was conducted from February 2017 to February 2019. Primers specific for T. gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and T. pallidum were designed using Primer3 software ( The primer sequences obtained were subjected to BLAST analysis using BLAST database. Synthetic DNA was obtained to use as positive control templates for all the six TORCH-S agents. The lower limit of the detection was performed using plasmid construct for each virus serially diluted from 10−1 to 10−9. Results: An in-house multiplex real-time PCR was standardized and validated in two cocktails for TORCH-S agents, cocktail-1 (HSV1, rubella, and T. gondii), and cocktail-2 (HSV2, CMV, and T. pallidum). The lower limit of the detection for HSV1, rubella, and Toxoplasma were 60.7 copies/10 μl input, 76.4 copies/10 μl input, and 34.4 copies/10 μl input and for HSV2, CMV, and T. pallidum were 80.8 copies/10 μl input, 166 copies/10 μl input, and 43.7 copies/10 μl input, respectively. Conclusion: TORCH-S infection is one of the significant reasons for irregular pregnant outcomes. It is absolutely important to screen TORCH-S infection for women who had the histories of abnormal pregnancies to prevent birth defects and perinatal complications. This multiplex real-time PCR assay provides a rapid, sensitive, and specific technique to detect these six TORCH-S agents.

Keywords: High-risk pregnancy, real-time polymerase chain reaction, Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2) and Treponema pallidum (syphilis) infections

How to cite this article:
Rajendiran P, Saravanan N, Ramamurthy M, Sankar S, Aruliah R, Nandagopal B, Sridharan G. Standardization of an in-house multiplex real-time polymerase chain reaction for the simultaneous detection of Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex Virus 1 and 2, and Treponema pallidum infection among pregnant women. Indian J Public Health 2021;65:369-74

How to cite this URL:
Rajendiran P, Saravanan N, Ramamurthy M, Sankar S, Aruliah R, Nandagopal B, Sridharan G. Standardization of an in-house multiplex real-time polymerase chain reaction for the simultaneous detection of Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex Virus 1 and 2, and Treponema pallidum infection among pregnant women. Indian J Public Health [serial online] 2021 [cited 2022 Jul 6];65:369-74. Available from:

   Introduction Top

TORCH-S is a medical acronym for a set of perinatal infections with known adverse impacts on fetal development and pregnancy outcomes. The full form of TORCH-S is Toxoplasma gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and Treponema pallidum (syphilis).

Toxoplasmosis is caused by the parasite T. gondii. Infected pregnant women are often presented with symptoms such as mild or asymptomatic, making the diagnosis difficult.[1] The organism is transmitted hematogenously to the placenta. When this occurs, an infection may be transmitted to the fetus transplacentally or during vaginal delivery.[2],[3] The clinical implications of infection due to toxoplasmosis in pregnant women could lead to spontaneous abortions, stillbirths, intrauterine growth retardation, preterm deliveries, or fetal damage.[4] Worldwide, 190,100 infants born with congenital toxoplasmosis.[5]

The clinical manifestations of rubella include a mild exanthema that is frequently accompanied by adenopathy and occasionally arthralgia. It can cause fetal death or congenital rubella syndrome (CRS) during the first trimester, which is characterized by multiple defects to cataracts, congenital heart defects, neurological problems, hepatomegaly, and splenomegaly.[6] It has been estimated that during 1996–2010; globally 105,000 infants with CRS were born every year, among them 38% of which were from India.[7]

Cytomegalovirus (CMV) is the most common cause of congenital infection and complicates approximately 1% of every single live birth.[8] Primary maternal CMV carries a 30%–40% risk of vertical transmission. Moreover, congenital CMV is a frequently identified viral cause of mental retardation and is the leading nongenetic cause of neurosensory hearing loss.[9],[10],[11]

Herpes simplex virus (HSV1 and HSV2) infections are transmitted from pregnant women to their neonates. It causes even death in infants and is associated with lifelong infection. HSV-1 and HSV-2 both can be responsible for neonatal, which can lead to spontaneous abortions, intrauterine growth retardation, preterm labor, and congenital and neonatal herpes infections.[12] The greatest risk of transmission to the fetus and the newborn occurs in case of an initial maternal infection contracted during the second half of pregnancy. Worldwide, herpes simplex virus infections are common, are transmitted from pregnant women to their neonates, and can cause infection or death.[13],[14]

Congenital syphilis, the infection caused by the spirochete T. pallidum, can be transmitted sexually or from mother to child in utero during the second or third trimesters of pregnancy.[15],[16] The most maternal syphilis infections are asymptomatic, but still result in poor pregnancy outcomes in more than 50% of cases.[17] Congenital syphilis could lead to spontaneous abortions, stillbirths, intrauterine growth retardation, preterm deliveries, or fetal damage. If the infection is untreated, it may even lead to complications such as early fetal loss, preterm birth, and low birth weight.[18]

The TORCH-S IgM assays have some limitations in their specificity producing up to 10% false positives. It has been reported that the routine practice of screening for IgM class antibodies during pregnancy may lead to numerous false-positive results, which can cause needless worry as well as unnecessary follow-up testing and treatment. Because of the crucial medical interventions required both for the mother and the child, it is important to have assays with extremely high sensitivity and specificity. In contrast to serological testing, the polymerase chain reaction (PCR) assay allows for earlier testing as viral genomic material seen in blood and body secretions of the infected mother, the PCR testing improves the specificity of the results.[19]

Serological assays which are the mainstay of diagnosis have several limitations. First, individuals may vary in their antibody response to the antigens used in the assay both temporarily and in magnitude. Second, antigenic cross-reactivity could be a problem causing false positives. Third, the IgM assays could be falsely negative in the presence of an excess of specific IgG. Furthermore, the rheumatoid factor could interfere while not using IgM capture format assays of rheumatoid factor absorbent for the pretreatment of sera. In contrast, genomic material will be present very early in infection and the entire duration of active pathogen replication. The choice of highly conserved specific sequences will overcome pathogen cross-detection by PCR. Hence, we standardized an in-house multiplex real-time PCR assay and validated using plasmid control. This assay can simultaneously detect the following 6 common TORCH-S agents: T. gondii, Rubella virus, cytomegalovirus, herpes simplex virus (1 and 2), and T. pallidum (syphilis) in cocktail-1 (HSV-1 rubella, and T. gondii) cocktail-2 (HSV2, T. pallidum, and CMV). In the recent years, the real-time PCR has improved the diagnostic of viral and bacterial infections, being a powerful tool for the detection and quantification of RNA or DNA. Real-time PCR is increasingly used in diagnostics due to its high sensitivity and good reproducibility. This study was approved for ethical clearance by Sri Narayani Hospital and Research Centre Ethical Committee (No: IEC/IRB No: 21/04/06/11, dated: 04/06/2011).

   Materials and Methods Top

This study was cross-sectional in nature and conducted from February 2017 to February 2019 in Sri Narayani Hospital and Research Centre (SNH and RC), Sripuram, Vellore, Tamil Nadu, India. The laboratory study was carried out in Sri Sakthi Amma Institute of Biomedical Research, a unit of SNHandRC.

Selection of target genes

The first step in the development process of an in-house multiplex real-time PCR assay is the choice of a nucleic acid target. A literature review often reveals which target is the most suitable for each particular assay. For TORCH-S, specific and conserved nucleic acid target sequence was selected.

Primer and probes design

Selection of the target sequence has been done, next to find potential primers and probes targeting regions of the corresponding sequence using Primer3 software ( Primer3 is one of the most widely used primer design software; it is a frequently updated and open-source project and used many web-based applications to develop useful functions for primer and probe designing. The Primer3 software is widely used for designing PCR primers, hybridization and sequencing primers.

Validation of primers and probes

The amplicon's specificity was confirmed using the BLAST program. This software searches different databases for sequence similarity and returns a collection of gapped alignments with links to complete database entries. Both the maximum identity and query coverage should be 100%. The “expectation value” (E-value) is a measure of statistical significance that is given to each alignment produced by BLAST. It is an indication of the likelihood of discovering the match by chance. The E-value is a commonly used metric for determining the likelihood of a biological connection. Smaller E-values indicate a higher probability of an underlying biological connection. Sequences having E-values of ≤0.01 are most often found to be homologous. Validated-Primer and probes were custom manufactured commercially by Eurofins Genomics (Germany) often shipped and received in a lyophilized state. Primers and probes were resuspended in TE (10 mMTris•Cl, 1 mM EDTA, pH 8.0) to provide a stock solution of 100 μM. The resuspended primers and probes were aliquoted in 0.6 ml tubes. This reduces the number of freeze/thaw cycles that the master primer aliquots go through and also reduces the chances of contaminating the primary source for the primers and probes.

Development of gene constructs for positive control and lower limit of detection

Synthetic DNA was obtained to use as positive control templates for all the six TORCH-S agents. A positive control is necessary, when amplifying a target sequence to confirm whether the primer set or primer–probe set works. The synthetic genes were designed such that the final synthetic gene length for all the constructs was 250 bp. The specific genomic region for each of the six TORCH-S agents was obtained commercially from Eurofins Genomics (Germany). The synthetic gene was obtained subcloned into a vector (vector: PCR 2.1, Cloning: TOPO-TA). These constructs were used to establish a lower limit of detection as well as to serve as positive control templates for all our real-time PCR assays. The nucleotide sequences of real-time primers used for two mixes are shown in [Table 1].
Table 1: Primers and probes information for real-time polymerase chain reaction target genes

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Evaluation and performance of real-time multiplex

The real-time PCR amplification was performed in multiplex for each agent as 25 μl reaction using QuantiTect a multiplex NR kit (Qiagen, Hamburg, Germany). Positive control (10 μl), forward and reverse primers (300 nM), and probe (200 nM) were used with initial polymerase activation (PCR) for 15 min at 95°C and followed by 45 cycles of denaturation at 94°C for 45 s. Annealing/extension was at 60°C for 75 s. Amplification and detection were performed on a real-time PCR system (Rotor-Gene Q) using the Taqman principle. The cutoff for real-time PCR endpoints was determined as amplification within the 40th cycle with a fluorescence intensity or more with a typical sigmoid amplification.[20],[21]

Determination of efficiency and limit of detection

Construction of a standard curve from a serial dilution of the plasmid control is the most effective way to evaluate assay performance. The standard curve gradient may be used to determine the assay's efficiency. The technical assay dynamic range may be determined from the same experiment by running a broad variety of sample concentrations and verifying that they reach a limiting dilution. [Figure 1] shows how to use a standard curve to determine an assay's technical dynamic range and efficiency. The plasmid construct was serially diluted 10-fold in TE buffer (pH 8.0) in the concentration ranging from 10−1 to 10−9. Each dilution was tested in triplicates by real-time PCR. Appropriate negative controls were used replacing the template with nuclease-free water and included as every third sample. The PCR runs were validated only if the controls were satisfactory. Amplification shown in the highest dilution in at least two replicates of the triplicates tested at each dilution was taken as the lower limit of detection as plasmid copies per microliter. The approximate number of plasmid copies per microliter of DNA suspension was thus established. The calculation of the plasmid copy number of the agents tested was done according to the standard methods.[22] The primers' specificity was determined by testing them with heterologous plasmids.
Figure 1: An example of high reproducibility and wide range of detection using a serial dilution of linearized plasmid.

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Multiplex standardization

Multiplex real-time PCR uses a different set of primer pairs in the same reaction for simultaneous amplification of multiple selected target regions. Standardization of multiplex real-time PCR assays was performed using plasmid controls obtained commercially from Eurofins Genomics (Germany); uniplex real-time PCR was performed to ensure the specificity of the primers and probes. In all channels, each pathogen plasmid control was detected only for the specific set of primers and probes.


To establish meaningful comparisons between various samples, the amplification efficiency of PCR reactions was evaluated. To assess the efficiency of amplification in a particular primer set, each virus included in the research was serially diluted from the original control, and a standard curve was created. The efficiency was calculated according to the following formula: E= (10 [−1/pendiente]) – 1.

Diagnostic of sensitivity and specificity

An assay of analytical sensitivity was performed in uniplex and multiplex reactions to evaluate the behavior of the assay in response to changes in the quantity of nucleic acids. For each virus included in the research, these tests were conducted in triplicate serial dilutions in base 10 (1 10−1 to 1 10−9) from the original control. To establish the starting concentration, all of the controls were measured.

   Results Top


Standardization of an in-house multiplex real-time PCR assays was performed using synthesized plasmid control. Initially, the assay was performed in uniplex to ensure the specificity of the primers and probes. The uniplex PCR assay result for each virus was detected only for the specific set of primers and probes used. The lowest Ct value for the T. gondii was 16.49, 17.07 for Rubella, 17.77 for HSV1, 18.28 for HSV2, and 16.97 for CMV, and the highest for T. pallidum was 18.65. (Multiplex PCRs were tested to verify that cross-reactions were avoided and that results were similar to those obtained for uniplex reactions). Amplification plots for cocktail-1 and cocktail-2 are shown in [Figure 2].
Figure 2: Amplification plot for cocktail 1 and cocktail 2: Amplification plot for HSV1 (ROX, orange) Rubella (Cy5, red) and Toxoplasma gondii (FAM, green) positive controls in cocktail 1 and Amplification plot for CMV (FAM, green), HSV2 (Cy5, red) and Treponema pallidum (ROX, orange) positive controls in cocktail 2.

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Efficiency was calculated for each virus in multiplex PCRs reactions, obtaining efficiencies of 90% for rubella, 99% for T. gondii, CMV, and T. pallidum obtained same efficiency 93% HSV1 and HSV2 virus showed the lowest efficiency (89%).

Determination of efficiency and limit of detection

The inhouse realtime PCR assay had a detection limit of 166 genome copies per 10 μl of PCR input for CMV, T. gondii show the lowest copy number 34.4 genome copies per 10 μl of PCR input. The copy numbers indicating high assay sensitivity to detect and also the low copy numbers. The lower limit of detection for TORCH-S agents was determined. The lower limit of detection established for the TORCH-S agents is shown in [Table 2].
Table 2: The lower limit of detection for Toxoplasma gondii, Rubella virus, cytomegalovirus, Herpes simplex agents was determined. The lower limit of detection established for the Toxoplasma gondii, Rubella virus, cytomegalovirus, Herpes simplex agents

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   Discussion Top

The multiplex real-time PCR assay has many advantages compared to other diagnostics methods, including speed, quantitative measurement, lower contamination rate, higher sensitivity, and higher specificity.[23] There are several methods that exist for the fast identification of viral infections. However, the molecular tests have shown excellent performance and may be an option for the diagnostic of routine within the laboratory.[24]

This research discusses the creation of six real-time multiplex PCRs that may be used in conjunction to identify six congenital infections. Using commercially produced plasmid controls, tests for lower limit of detection, efficiency, specificity, and sensitivity were evaluated, establishing the robustness of the standardized multiplex real-time PCR.

The technologies, uniplex and multiplex real-time PCR, were shown to have high sensitivity. For each pathogen, the lowest dilution suggests signal for the multiplex system spans from Ct 16.49 to Ct 18.24. The results were fairly comparable for both kinds of tests, but the Ct values in the multiplex PCR assays were greater. When multiple primers and probes are employed in the same mixture, this situation may be caused by a kinetic reaction.

The specificity tests revealed that the primers and probes are specific for the pathogens studied (Cocktail1 and 2), and no crossreactions were observed when genetic material from technically in each cycle the number of copies of genetic material doubles in realtime PCR. The Ct value for each dilution (1:10) obtained around 3.32.[25] By graphing the Ct values against dilution, the slope (m) reflects this value, and the line equation is calculated. It is a major problem since the PCR reaction efficiency should be between 90 and 100 percent (slope between 3.32 and 3.6).[21] If the efficiency is 100%, Ct values of 3.32 shows in each dilution for each real-time PCR cycle. When the slope reaches <−3.6, the effectiveness of the real-time PCR starts to decline. Furthermore, the value of R2 for a standard curve indicates how well the experimental data fits the regression line, i.e., how linear the data are. As a result, R2 should ideally be more than 0.9925. The slopes of various pathogens for multiplex real-time PCRs in our research vary from 3.34 and 3.63.

The efficiency of PCR amplification is often expressed as a percentage, i.e., the proportion of amplified genetic material in each cycle, with most pathogens having efficiencies around 90%. The lowest efficiency was found for HSV1 and HSV2 virus with a percentage of amplification of genetic material in each cycle of 89%. In contrast, higher efficiency was found for T. gondii with a value of 99%

The PCR efficiency is sufficient, since for 100% efficiency, with efficiencies of 90% achieved in our research. A molecular interaction in PCR efficiency makes a significant variation in the amount of end product. This scenario requires a large number of cycles to detect a certain quantity of genetic material.[26] Aside from this variation, the findings show that multiplex real-time PCR has excellent agreement between results for each run, ensuring reliable results.

The efficiency of real-time PCR varies according to assay performance; intramolecular interactions decrease real-time PCR efficiency; competing reactions and reagent quantity are additional factors in efficiency variance.

   Conclusion Top

The in-house multiplex real-time PCR assays used in this research performed well in detecting six TORCH-S agents closely linked with congenital infections. The TORCH-S multiplex real-time PCR assay has benefits in that it does not need post-PCR processing and may be utilized in rapid diagnostic procedures to identify TORCH-S infective agents.


This work was funded by the Indian Council of Medical Research (ICMR), Government of India (Grant ID: VIR/79/2013/ECD-1).

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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