Perspective Chapter: Epstein-Barr Virus – Emphasis on Diagnostic Approaches

Abstract

Classical methods (morphological, immunomorphological, virological, and serological) such as microscopic analysis, virus culture, western blot, and enzyme-linked immunosorbent assay (ELISA) are used to diagnose Epstein-Barr virus (EBV) infections. All the above methods are time-consuming and unusable for quick and accurate diagnosis of EBV, and the low sensitivity of these methods sometimes causes a delay in the start of treatment. Rapid development steps in molecular biology techniques have profoundly affected the detection of viral agents. Molecular methods can be classified into three main groups: (1) target amplification methods, (2) probe amplification methods, and (3) signal amplification methods. The most considerable and practical group of techniques is target amplification methods. In this category, valuable and important techniques include polymerase chain reaction (PCR), multiplex PCR, reverse transcriptase PCR (RT-PCR), nested PCR, immuno-PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), and loop-mediated isothermal amplification (LAMP). In nucleic acid amplification systems in laboratory conditions, the target molecule is replicated in large numbers using enzymes to the extent that the product can be revealed by methods such as gel electrophoresis. The first and perhaps the most important and best system in which the target molecule increases in number is the PCR technique. In terms of scientific principles, this technique is very similar to DNA replication and is derived from it.

1. Introduction

Epstein-Barr virus (EBV), a human γ₁-herpesvirus, also known as human herpesvirus 4 (HHV-4), is an oncogenic virus. EBV infects most of the population worldwide and establishes a persistent lifelong, mostly asymptomatic infection in them [1]. The envelope structure of EBV, like the herpesvirus subfamilies, contains a protein tegument between the nucleocapsid and the envelope and an outer envelope with external virus-encoded glycoprotein spikes. The envelope typically acquires its final envelope in various cytoplasmic compartments, such as the trans-Golgi network (TGN) and endosomes, before their secretion into the extracellular space creates a toroid-shaped protein core [1, 2]. The genome comprises linear double-stranded DNA, approximately 172 kb, with more than 85 protein-coding genes [3, 4]. EBV has two main types, EBV-1 and EBV-2; they differ in sequence from the genes encoding the EBV nuclear antigens (EBNA-2, EBNA-3A/3, EBNA-3B/4, and EBNA-3C/6) [2].

Three distinct latent viral gene expression patterns can be observed in EBV-infected tissues. Type I latency refers to the minimal latent viral gene expression spectrum, namely the EBER and EBNA1 transcripts and the latent membrane protein (LMP2A). This pattern is found in circulating lymphocytes of healthy virus carriers and is also characteristic of Burkitt lymphoma and gastric carcinoma. Type II latency, further characterized by co-expression of LMP1 and LMP2B, is seen in Hodgkin’s disease, T-cell lymphoma, and nasopharyngeal carcinoma, all of which tend to occur in hosts with immunity. Type III latency is the totality of latent viral gene expression, transiently found in acute infectious mononucleosis and EBV-induced lymphocyte proliferation in animals’ immunocompromised hosts. Viral genes expressed at type III latency include all EBNAs (1, 2, 3A, 3B, 3C, LP), LMP (1, 2A, 2B), and EBER [5].

2. Etiology

The primary EBV infection has a transient viremia followed by an acute immune response. The virus can persist in a human host for life, particularly in teenagers and young adults. It is achieved by effectively evading the immune system through latent B-cell infection while also being able to replicate and detach from the oral mucosa. The virus is usually transmitted by infected saliva. It infects the epithelium of the pharynx, salivary glands, and parotid glands. It can also be transmitted through blood transfusions and bone marrow transplants [6, 7]. Common clinical symptoms of this disease include fever, sore throat, cervical lymphadenopathy, mild hepatitis, and atypical lymphocytosis [6]. EBV is the causative agent of infectious mononucleosis and is associated with 1% of tumors worldwide, such as a variety of benign and malignant lesions including oral hairy leukoplakia, inflammatory pseudotumor, post-implantation lymphoproliferative disorder (PTLD), Burkitt lymphoma (BL), Hodgkin’s disease (HL), non-Hodgkin’s lymphoma (NHL), nasopharyngeal carcinoma (NPC), and gastric cancer EBV-associated thickening (EBVaGC) [5].

In most instances, the clinical diagnosis is confirmed by examination of the peripheral blood and serologic evaluation without needing a lymph node biopsy [8]. In a complete blood count (CBC) analysis, white blood cells are usually elevated and reach 10,000–20,000/μL by the second or third week. Lymphocytosis often occurs in more than 10% of atypical lymphocytes, in which CD8+ cytotoxic T lymphocytes predominate. Mild leukopenia and thrombocytopenia were observed during the first month of illness. Liver function is impaired in more than 90% of cases, and serum bilirubin levels are elevated in 40% of patients. Serum aminotransferase and alkaline phosphatase levels are usually elevated [6].

3. Laboratory diagnosis methods

Laboratory detection of EBV is accomplished in several ways, and recent advances have focused on molecular analysis of viral DNA and RNA. EBV detection methods are diverse and can be divided into two categories: (1) Classical methods and (2) Novel and molecular methods. Classical methods include morphological, immunological, virological, and serological methods. The rapid and sensitive detection capabilities of nucleic acid amplification techniques make these methods a new and attractive tool for medical diagnostic laboratories, such as rapid verification of EBV. In molecular methods, the genome of EBV can be identified directly and rapidly [9].

3.1 Classical techniques

3.1.1 Electron microscope

Electron microscopy (EM) is an extraordinary technique to provide high-resolution images of viral specimens and the structure of whole virions [9]. EBV was first identified by electron microscopy of cultured Burkitt lymphoma cells. EBV nuclear antigen 1 (EBNA1) is a virus-encoded DNA-binding protein required to maintain chromosomal expression during latent infection and consistent expression in all EBV tumors. EBNA1 can be used as a marker to detect EBV by electron microscopy. Rapid advances in cryogenic transmission electron microscopy (cryo-TEM) can provide high-resolution images without chemical fixation and be applied to chilled samples at cryogenic temperatures. The atomic structure of EBNA1 can be determined by Cryo-TEM [10]. This technique is impractical for routine clinical us [9].

3.1.2 Histological and cytological methods

EBV is most commonly associated with infectious mononucleosis. However, an estimated 1% of tumors, including lymphoid, epithelial, and mesenchymal proliferative tumors, are associated with EBV infection [11]. EBV host cells are epithelial cells, lymphocytes, and muscle cells. Like other herpes viruses, EBV has two pathophysiologic stages, lytic and latent phase [7]. In primary EBV infection, B cells are the cellular targets of EBV, and latent EBV infection persists for life in B cells and nasopharyngeal cells [11]. Microscopically, nodes and other lymphoid organs affected by infectious mononucleosis can be confused with malignant lymphoma [8]. Lymph nodes affected by EBV show partial architectural effacement due to marked sinusoidal and capsular/extranodal infiltration by Immunoblasts that often have Reed-Sternberg-like features and atypia, in addition to follicular hyperplasia with ragged or mottled edges. Lymphoid follicles have tingible body macrophages and marked mitotic activity [12]. Early infections have prominent monocytoid B cell reactions and no epithelioid cells [13].

Immunohistochemistry (IHC) is a powerful technique in cytopathology that exploits specific binding between antibodies and antigens to detect and localize specific antigens in cells and tissues. The IHC method includes enzymes that cause the conversion of colorless substrates (antigens) into colored insoluble substances, which are deposited at the site of the antigens. Subsequently, a standard optical microscope can determine the state of the stained cell or tissue. The staining of essential EBV latency proteins, such as LMP-1, LMP-2A, EBNA-1, and − 2 in tumor biopsies, is used to confirm the presence of the virus and distinguish between EBV-associated and non-EBV-associated tumors [4].

3.1.3 Viral culture

The virus culture method is one of the most sensitive methods in the laboratory. Cell culture is one of the most commonly used methods for diagnosing Herpesviridae. This method is considered the gold standard in comparison with many diagnostic methods. Due to its slowness, low sensitivity, high tendency to contamination, and potential for virus release in the laboratory, accurate virus culture is essential, and serological and molecular methods are preferred [14].

3.1.4 Serological diagnosis methods

Antibodies are considered the main and most powerful biosensors in nature. Various diagnostic tests are developed based on antibodies in biotechnology. Immunoassays for antigen concentration have high specificity and sensitivity and have become standard research and clinical practice methods [9].

EBV antibodies are directed against the lytic and latent phase antigens in the humoral response. There are three main types of antibodies against EBV antigens [4], which are as follows:

1. Epstein-Barr virus viral-capsid antigen antibody (IgM): Anti-VCA IgM appears early in EBV infection and usually resolves within 4 to 6 weeks. Anti-VCA IgG appears during the acute phase of EBV infection, peaks 2–4 weeks after onset, subsides, and persists for the rest of a person’s life [4].

2. Anti-Epstein Barr virus (IgG) early antigen: Anti-EA IgG appears during the acute phase of the disease and usually drops to undetectable levels after 3–6 months. In many people, detecting antibodies to EA is a sign of an active infection. EA is usually expressed in the early stages of lytic transcription [4].

3. Epstein-Barr virus antigen (EBNA): Anti-EBNA antibodies, as determined by standard immunofluorescence, do not appear during the acute phase of EBV infection but develop slowly 2 to 4 months after the onset of symptoms and persist for the remainder of the patient’s life. EBNA-1 IgG antibodies persist for life. However, all individuals do not produce EBNA-1 IgG antibodies. Other EBNA enzyme immunoassays may give false positives [4].

Three methods serve as the first choice in serological methods: Indirect Fluorescent Antibody (IFA) Assay, which is still the “gold standard” method; different enzyme immunoassay (EIA) techniques, including solid-phase enzyme-linked immunosorbent assays (ELISA) and related methods such as luminescence-based detection of anti-EBV antibodies with antigen-coated beads, and Western blot analysis [15].

In EBV-associated tumors and immunocompromised patients, antibody detection may not be necessary. Thirty-two antibody types can be generated by EBV, which can cause a high potential for misinterpretation and remains challenging for physicians to identify. However, in addition to monitoring the patient to assess for any changes in the antibody profile, it is also helpful to perform other laboratory tests as an additional precaution. There are different methods for detecting EBV antibodies in serology with various advantages and limitations [16]. Related methods include anti-EBV antibody luminescence by antigen-coated beads and Western blot analysis. Detection of viral antigens is more suitable for small laboratories than for viral cultures. This test can also be done in laboratories located in remote areas. The sensitivity of this test is sometimes higher or lower than culture. This test has high specificity in diagnosing HSV virus so that it can differentiate between HSV-1 and HSV-2, EBV, and VZV. This test is performed by immunofluorescence staining or monoclonal antibody [15].

3.1.4.1 Monospot test

The serological test for EBV-specific antibodies is considered a gold detection standard, and the monoclonal or mononucleosis tests are most commonly used. The monospot method is a latex agglutination test that uses equine erythrocytes as the primary substrate and tests for specific heterophile antibodies produced by the human immune system in response to EBV infection. The exposure to equine Red Blood Cells may cause the sample to bunch up when these antibodies are found in blood samples from patients, indicating a positive agglutination reaction. The sensitivity ranges from 70 to 90%. The antibodies detected by monospot can be caused by conditions other than infectious mononucleosis. Additionally, studies have shown that monospot produces both false-positive and negative results. Interpretation of EBV antibody tests requires familiarity with these tests and access to the patient’s clinical information [15].

3.1.4.2 Direct fluorescent antibody

In the direct fluorescent antibody (DFA) method, also known as direct immunofluorescence, a monoclonal antibody directed against a unique antigen on the virus particle is conjugated to a fluorescent marker that can be seen with a fluorescent microscope to detect EBV in cellular smears directly. DFA staining confirms and validates smears rapidly added to cell culture in the DFA method. It is essential to state that high-quality samples should be obtained for this test, in which case its sensitivity, especially in early infections, may be more than 90% [9].

3.1.4.3 Neutralization test

The virus neutralization test detects antibodies capable of neutralizing the infectivity of the virus. Serial serum dilutions are mixed with a viable viral reference strain and incubated to allow any antibodies present to bind to the antigen and neutralize the virus, diluting into the cells. The presence or absence of viral growth is observed. The principle of the test is that antibodies prevent the growth of the virus in tissue culture. The most important advantage of this test is virus protection. One of the limitations of this method is that it can only be performed in Ref. laboratories, and neutralizing antibodies must be present in a specific titer to interpret positive results [9].

3.1.4.4 Immunofluorescence test

Immunofluorescence test (IFA) is a microscopic standard virologic technique to identify the presence of antibodies by their specific ability to react with expressed viral antigens in infected cells. Bound antibodies are visualized by fluorescence microscope via incubation with a fluorescently labeled antihuman antibody. The principle of this test is that the IgG or IgM antibodies attached to the points of the virus-infected cells are detected by the secondary antibodies labeled on the slide. The advantage of the test is that it can yield high antibody titers in a short time. One of the disadvantages of the test is that it is done manually, and receiving the test results is done subjectively [17].

3.1.4.5 ELISA

In situ hybridization has long been considered the gold standard for detecting EBV viral load assays. It is now adopted for clinical evaluation of tumor burden in affected patients [5]. The principle of the test is a plaque assay technique in which solid phase-bound virus-specific antibodies are detected by labeling anti-IgM and anti-IgG secondary antibodies. ELISAs can be performed with several modifications to the basic procedure: direct, indirect, sandwich, or competitive. The ELISA begins with the coating step, in which the first layer, including the target antigen, is coated onto a 96-well polystyrene plate. It is followed by a blocking step where all unlinked pages are coated with a blocking agent. After a series of washes, the plate is incubated with enzyme-conjugated antibodies. The detection antibodies are usually labeled with alkaline phosphatase (AP) or horseradish peroxidase (HRP). Another washing step will remove any unbound antibodies. Then, a substrate is added, generating a colorimetric signal. Finally, the plate is read by microplate equipment [9]. This method is quick, sensitive, automated, and commercially available. The limitation of this test is false positives and false negatives. IgG and IgM antibodies confirm a positive result [18].

The ELISA method was developed for the epidemiological screening and diagnosis of EBV. ELISA was accomplished with antibody function against latent phase EBV (EBNA1), early EBV (p47/54), and two late proteins (gp350/220 and VCA-p18) [19].

3.1.4.6 Chemiluminescence

Today, many non-radioactive techniques have been used as diagnostic alternatives in immunoassays. Chemiluminescence (CL) or luminescence (L) method is an extensive technique. This technique is used worldwide in various measurements and models. Chemiluminescence describes light emission due to specific chemical reactions that produce large amounts of energy lost in photons when the electronically excited product molecules transition to the ground state. They are stable and are usually caused by oxidation reactions. This feature minimizes background light and is theoretically zero. In a very short time, all the emitted photons from the luminescent compounds can be generated, increasing the specificity of the diagnosis. Immunofluorescence assays provide several times higher sensitivity than radioisotope and fluorescence immunoassays [9, 20].

The most common chemiluminescent compounds are acridinium esters and luminol derivatives, which are highly sensitive. They can also be conjugated to antigens and antibodies by standard methods. The identification method is almost simple and is done by adding a hydroxide and hydronine peroxide solution. This reaction produces a sudden flash of light. A specially designed oligonucleotide probe (BamHIW) directly labeled with alkaline phosphatase (AP) and a susceptible CDP star chemiluminescence substrate for AP was used in the hybridization assay [21].

3.1.4.7 Western blot

Western blot is a reference method for the detection of virus antibodies. Various Western blot techniques have been established for confirmation of screening test results. This test is a sensitive and specific serological method that allows a definitive virus diagnosis. This technique uses the directed antibodies against specific viral proteins visible by electrophoresis. Although this technique is exact, it requires the skill and expertise of the technician. The basic technique involves the separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [18]. It transfers onto a support membrane such as nitrocellulose or polyvinylidene difluoride (PVDF), followed by immunostaining with specific antibody reagents [22].

A monoclonal antibody directed against the diffuse component of the primary antigen (anti-EA-D) in various human lymphoblastic cell lines was used to detect EBV. Examples of these include methods with EBV-transfected cells and cloning assays with recombinant antigens, such as p72 (EBNA-1), p18 (VCA), p23 (VCA), p54 (EA), and p138 (EA). The p18 VCA antigen is considered a surrogate marker for the absence of EBNA-1 IgG because IgG p18 is mainly produced late in infection. Recombinant EBV-specific antigens are superior to lysate spots because of the potential antibodies that react with anti-cell material commonly present in patients. Monocytes did not affect the results [16].

3.1.4.8 Southern blot

Southern blot is a commonly used method in molecular biology to detect specific DNA sequences in samples. In the Southern blot method, DNA fragments separated in an agarose gel are transferred to a membrane and fixed. After translocation of the fragments in the gel, the DNAs are denatured with alkali. Membrane-bound single-stranded DNA can bind to complementary sequences in this method. Then, DNA fragments are transferred to the membrane. The transfer between the gel and the contact membrane is accomplished by electrophoresis or capillary tension, which causes the buffer to flow from the gel to the membrane, and the DNA fragments migrate with the buffer. They are trapped in nitrocellulose or nylon film. After DNA transfer, it is necessary to stabilize the DNA fragments on the membrane. Heating (80°C) and UV irradiation are two main available methods for stabilization. After immobilization, the membrane is placed in a solution containing a labeled single-stranded nucleic acid, usually with radioactivity, RNA, or single-stranded DNA [9].

The labeled nucleic acid or probe can hybridize with complementary sequences present in the membrane. The interaction between the single-stranded probe and its complementary sequence binds the probe to the membrane by non-covalent hydrogen bonds that naturally hold the two DNA strands together. This membrane is then washed to remove probes with nonspecific binding and exposed to X-ray film to show specific binding [9].

3.1.4.9 Aptamer

Aptamers are small single-stranded RNA or DNA oligonucleotides (30–70 nucleotides) with high affinity for binding to a specific target. This property of the aptamer is comparable to the function of antibodies against antigens. Isolation of aptamers is accomplished through an in vitro process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This process generates sequences with high affinity for the target [9]. Aptamer was used to detect EBV in nasopharyngeal carcinoma [23].

3.1.4.10 Flow cytometry

Flow cytometry is a technology that provides rapid multi-parametric analysis of single cells in solution through the flow cell in the form of a laminar flow in the hydrodynamic concentration zone and front of the laser light, which in terms of physical characteristics such as size, shape, and complexity of the nucleus, as well as in terms of the indicators in the surface of the cytoplasmic membrane and the nucleus are examined utilizing monoclonal and polyclonal antibodies labeled with fluorescent substances. Novel flow cytometry with a phosphorescence method has been designed to detect 60 biological factors simultaneously. This device can detect the presence of agents with optical and electrical methods at a speed of 1000 cells per second with continuous sampling. To detect microscopic particles such as viruses and proteins, they use tiny and unique beads whose surface is covered with antigens against biological agents [9, 24]. To diagnose EBV infection and EBV-associated disease, the EBV load can be measured by flow cytometry [25].

3.1.4.11 Fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) is a cytological technique. FISH can detect specific sites of specific DNA sequences in metaphase or interphase cells using fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. In biopsy tissues, molecular detection of EBV-encoded RNA transcripts by in situ hybridization remains the gold standard for proving that a histopathological lesion is related to EBV [9].

Epstein Barr encoded RNA (EBER) in situ hybridization (EISH) assay is the gold standard for detecting and diagnosing EBV infection. The EISH technique employs nucleic acid probes, either labeled or unlabeled, which can hybridize with EBER on paraffin sections of EBV-infected tissues [4].

All methods mentioned above are expensive, time-consuming, and unusable for quick and accurate diagnosis of EBV. The low sensitivity of these methods sometimes causes a delay in the start of treatment. The rapid development stages of molecular biology techniques have profoundly impacted diagnostic applications, especially in detecting infectious agents and other applications (Table 1).

Parameter	Viral culture	Serological methods	Non- amplification probes	Gene amplification methods
Speed	+	+++	++	++/+++
Sensitivity	+++	++	++	++++
Specificity	+++	++	+++	++++
Quantifiability	++	++	+	+++
Easy to use	+	+	+	++/+++

Table 1.

Comparison of classical and molecular techniques.

(+ = weak, ++ = medium, +++ = good, ++++ = strong)

3.2 Molecular methods

The invention of nucleic acid amplification techniques has made these methods increasingly practical in various laboratories, especially clinical laboratories [18]. Molecular diagnostic tests have been able to quantify viral genomic sequences in clinical samples reliably. Nucleic acid amplification methods in laboratory conditions can be divided into three main groups: (1) Target Amplification methods, (2) Probe amplification methods, and (3) Signal amplification methods were classified. Target amplification is the most essential and practical technique group. Molecular testing is increasingly important in diagnosing and monitoring patients affected by these diseases [26].

In the target amplification systems group, valuable and important techniques such as polymerase chain reaction (PCR), multiplex PCR, reverse transcriptase PCR (RT-PCR), nested PCR, immuno-PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), and loop-mediated isothermal amplification (LAMP). In nucleic acid amplification systems in laboratory conditions, the target molecule (DNA or RNA) is replicated in large numbers using enzymes to the extent that the product can be revealed by methods such as gel electrophoresis. The first and perhaps the most important and best system in which the target molecule increases in number is the PCR technique. In terms of scientific principles, this technique is very similar to in vivo DNA replication and is derived from it. The probe amplification methods include ligase chain reaction (LCR), strand displacement amplification (SDA), and Qβ replicase, and the signal amplification methods include branched DNA amplification, hybrid capture assays, cleavage-based amplification, and cycling probe [18]. This chapter only discusses target amplification methods to detect EBV.

3.2.1 Polymerase chain reaction

The in vitro polymerase chain reaction (PCR) method is the primary and most important system for amplifying the target molecule. PCR allows amplification of DNA sequences and can be a convenient method for gene identification and quantification. After Kerry Mullis invented the PCR method in 1985, it quickly became used in all molecular diagnostic and research laboratories worldwide, creating a revolution in molecular biology. In PCR, nucleic acid amplification uses Taq DNA polymerase enzyme and two specific primers using thermocycler [18].

The basis of the PCR technique is the enzymatic synthesis of a strand of DNA produced in several thermal cycles. The principles and rules based on the PCR-reaction method are similar to those governing replication in living cells. The main components of the PCR-reaction template strand are primer, buffer, Taq DNA polymerase, primer pair, dNTP (dATP, dCTP, dGTP, dTTP), and Mg²⁺ [9].

The PCR method contains three stages. In the initiation step, the mixture heats to 94°C by using a thermocycler, the double-stranded DNA template strand to the point where the strands start denaturing, and the hydrogen bonds are broken between the nucleotide base pairs, which is called the denaturation step. In the cyclic process of PCR, if the temperature is reduced to 40 to 65°C, in this case, the oligonucleotide sequences of 18 to 25 nucleotides can be attached to the separate and single-stranded DNA strands in the solution, which is called the annealing step. After the oligonucleotide sequences are attached to the target region on the DNA strand, if the environment’s temperature reaches 72°C, the amplification occurs in the target region in the presence of the DNA polymerase enzyme, which is called extension. In a cyclic and repeatable process, the above three steps are repeated during 25 to 30 thermal cycles in the PCR technique. Therefore, millions of copies can be produced from a minimal amount of target DNA [26].

The PCR technique can quickly identify EBV with high sensitivity and specificity, which is significant in determining the prognosis and effectiveness of treatments. Conventional PCR was performed with a set of reports of EBNA-1 oligonucleotide primers and using Taq DNA polymerase [27].

Molecular diagnostic tests have been developed to reliably quantify the number of genomic sequences in clinical samples. Modified methods have been created for more accurate use of PCR techniques in diagnosis [18]. Among the molecular diagnostic methods, different types of PCR methods, such as reverse transcription PCR (RT-PCR), nested PCR, multiplex PCR, and real-time PCR, are the most widely used. The real-time method is superior to conventional PCR in terms of reducing the possibility of contamination, speed, specificity, quantitative measurement, and easy standardization [28].

3.2.2 Multiplex PCR

One of the modified methods of conventional PCR is the multiplex PCR method. This method amplifies more than one target sequence using multiple primer pairs in a reaction mixture [9]. Thus, the same PCR reaction includes two or more primer sets to amplify different target sequences in a clinical specimen that can be amplified in a single tube [29].

Primers in multiplex reactions should be carefully selected for similar annealing temperatures. These primers should not be complementary to each other. This technique detects cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in preserved paraffin sections of lung tissue from immunocompromised patients [30].

3.2.3 RT-PCR

Reverse transcription polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique for viral agent identification. The RNA template can be used as a target in RT-PCR. This target RNA is first converted to cDNA using reverse transcriptase (RT) enzymes [18, 28].

The RT enzyme, a thermostable DNA polymerase (called Tth), isolated from a thermophilic bacterium (Thermos thermophiles), has reverse transcriptional activity in the presence of Mn²⁺ ions. This enzyme can generate DNA (cDNA) from RNA at 72°C in the presence of Mn²⁺ [28]. RT-PCR was developed for Epstein-Barr early RNA (EBER) to quantify EBV load rapidly [31].

3.2.4 Nested-PCR

Another PCR method is nested polymerase chain reaction (Nested-PCR), which is used as needed to increase the sensitivity and specificity of PCR. Nested PCR typically consists of two sequential amplification reactions, each using a different pair of primers. This method first amplifies the target gene by an external primer pair for 15–30 cycles. Then, the resulting PCR product was transferred to another tube, served as a template, and using the primer pair inside, the second PCR step was performed for 15–40 cycles. PCR products are separated by gel electrophoresis [28, 32]. The detection limit of conventional PCR methods is less than 50 to 100 copies per milliliter of sample. The mentioned method provides very high specificity for gene amplification due to using a more significant number of primers. However, because this method is performed in two steps, the possibility of product contamination during moving from the first PCR step to the second step results in a false-positive reaction. Therefore, trained personnel are necessary to prevent contamination [28].

Nested PCR was performed with a reported set of EBNA-1 oligonucleotide primers; (5′-GTAGAAGGCCATTTTTCCAC-3′) and (5′-CTCCATCGTCAAAGCTGCA-3′) as the outer primers and (5′-AGATGACCCAGGAGAAGGCCCAAGC-3′) and (5′-CAAAGGGGAGACGACTCAATGGTGT-3′) as the inner primers and using Taq DNA polymerase [33].

3.2.5 Immuno-PCR

The immuno-PCR method is a developed quantitative method using a specific DNA molecule as a marker. qIPCR detects antigens using specific antibodies labeled with double-stranded DNA, and the marker is used for signal generation by quantitative PCR [34]. The initial qIPCR protocol was the opposite of the ELISA protocol. In ELISA, the Ab-Ag combination is used, and the enzyme substrate is added freely and dispersed, but in the qIPCR technique, the enzyme substrate is connected to the antibody with the same DNA pattern and the enzyme is added later by strengthening the power of LOD (detection range) increases between 100 and 1000 times [28].

This method is a combination of ELISA and PCR techniques so that it can detect protein antigens according to the ELISA method. On the other hand, using the Real-Time PCR method can specifically identify only the sequence related to the target antigen and increase this sequence during multiple PCR cycles for more accurate Detection. As a result, due to its very high power in detecting protein antigens and protein antibodies, this method is mainly used to implement bacterial and viral antigens [9].

3.2.6 Real-time PCR

PCR is considered an accurate and fastest diagnostic method in many scientific fields due to its development as a qualitative and quantitative technique. Different modifications in execution and detection mechanisms, such as labeled material, are applied in PCR for more convenient use. Real-time PCR is the meeting point of innovations in the thermocycler field, different primer developments, and blending hybrid methods with amplification techniques. A new generation of thermal cyclers with fluorescence measurement systems has recently been introduced to the market, allowing continuous control of the fluorescence characteristics of the PCR product during assembly. In these systems, a probe or hybrid probe labeled with a fluorescent dye at the “5′” or “3′” ends is used, allowing continuous monitoring of the PCR product without the need to separate them by other means—methods such as agarose or polyacrylamide gel electrophoresis [9, 28].

In real-time PCR, no operations are required after the amplification step. Therefore, these measurement methods are called closed or homogeneous systems. One of the advantages of homogenous systems is the reduction of contamination as a major pest of the molecular method and the ability to make precise measurements. In the PCR method, the product tracking is done at the endpoint, while in the real-time method, the product tracking is done cyclically while the reaction is ongoing. Since the nucleic acids are amplified and the detection steps are carried out in a closed environment, this dramatically reduces the risk of environmental contamination and subsequent reactions compared with traditional PCR methods. Rapid tests eliminate the step of tracking the product after PCR, observing the reaction at any time and stopping it at any time, high sensitivity and specificity, performing the quantitative reaction, and getting the exact number of sets of genes and prototypes in real-time PCR method is revolutionizing It has become essential in molecular detection of viral agents [18, 28]. Real-time PCR was developed for the diagnosis of the BALF5 gene encoding the EBV DNA polymerase [35].

3.2.7 Nucleic acid sequence-based amplification

Nucleic acid sequence-based amplification (NASBA) is one of the isothermal amplification methods without the need for a thermocycler. It consists of a set of different enzymatic reactions in which RNA with a specific sequence is used as a template for the reverse transcription reaction. RNA polymerase T7 enzyme causes DNA synthesis from RNA followed by hydrolysis of RNA bound to DNA. After creating cDNA from the target RNA and digesting the strand attached to DNA, the second primer is attached to the newly synthesized cDNA strand and the action of the polymerase enzyme forms double-stranded DNA. NASBA and 3SR methods have very high sensitivity. They can amplify less than ten copies per milliliter of the target nucleic acid within less than an hour. Still, their low specificity in selecting the target sequence requires precise amplification tools and complex product detection methods [28].

NASBA was developed for the direct detection of EBV transcripts encoding EBNA1, LMP1, and 2, and BamHIA rightward frame 1 (BARF1) and the noncoding EBV early RNA 1 (EBER1) [36].

3.2.8 Loop-mediated isothermal amplification

In 2000, Notomi et al. reported a new generation of gene amplification techniques, Loop-mediated isothermal Amplification (LAMP), for detecting microbial and viral infections. In general, the LAMP method can have potential applications for clinical diagnosis and monitoring of infectious diseases in developing countries due to its simplicity, high amplification speed, and easy detection [18]. This method amplifies the target nucleic acid by the Bst DNA polymerase enzyme and six specific primers (FIP, BIP, F3, B3, LF, LB) that recognize eight regions in the target sequence. Bst DNA polymerase enzyme is a modified gene product of Bacillus stearothermophilus-derived DNA polymerase with a molecular weight of 67 kilodaltons. This enzyme has 5′ to 3′ polymerase activity and 3′ to 5′ exonuclease activity. Also, at the same time as polymerization, it can separate two DNA strands and self-cycle DNA synthesis. The optimal temperature for activity of the enzyme is 60–65°C (especially 63°C). This enzyme cannot be used for PCR and sequencing with temperature cycles due to its inactivation at a temperature higher than 70°C. However, it can be used in isothermal DNA Amplification, especially the amplification of complex regions such as repetitive sequences, GC-rich regions, and secondary structures. The LAMP method does not require a thermocycler device. For induction of optimal temperature (62–65°C), a water bath or heat block can be efficiently used [28].

The low cost of the analysis equipment is the most important advantage of the LAMP method, especially for developing countries with limited financial resources. The unique structure of LAMP primers and their number of copies provide more starting points for DNA polymerase and lead to increased efficiency and speed of replication. In this process, the target DNA increases by 10⁹–10¹⁰ copies (considerable product) in 15–90 minutes. Gel electrophoresis can be used to detect LAMP products, like PCR products. LAMP leads to more significant production than PCR. Thus, there is no need to use electrophoresis and the carcinogenic dye, ethidium bromide. For observation of amplification results, fluorescent dyes such as SYBR Green, Calcein, Picogreen, Gold Nanoparticle, and Hydroxy Naphthol Blue can be used via DNA binding and accumulation of color side products.

In addition, along with the original product in the LAMP reaction, many side products, such as pyrophosphate, are also created. The reaction of pyrophosphate with magnesium produces white precipitation of magnesium pyrophosphate. Therefore, the mixture becomes cloudy at the end of the LAMP reaction. The amount of precipitation is associated with the amount of DNA products. So, a linear relationship exists between the amount of DNA produced and the turbidity. Thus, the number of products can be measured by assessing the change in turbidity with a spectrophotometer and drawing a standard curve. The number of products and the number of gene copies can be quantitatively measured by the real-time method or integrated with the reverse transcription method if RNA is the target by adding reverse transcriptase enzyme to the reaction mixture, RNA is amplified as a template, and by eliminating the additional step of cDNA synthesis, it saves time and money. Another essential advantage of the LAMP reaction is that it is less affected by the present inhibitors in clinical samples than the PCR reaction. Also, unlike PCR, extracting the target nucleic acid is unnecessary and can be performed directly on clinical samples with lower sensitivity. Using the LAMP method, it is possible to detect the number of low copies (less than ten copies per milliliter) of DNA or RNA in the sample. In most of the conducted research, the sensitivity of the LAMP method is similar to nested PCR. It is 10–100 times more sensitive than standard PCR. The LAMP technique was used to determine HSV 1,2 and EBV [37, 38].

Acknowledgments

The author thanks Professor. Mohammad Hassan Shahhosseini, Dr. Hadi Zare Zardini, Professor. Seyed Hossein Hekmatimoghaddam, Dr. Fatemeh Pourrajab, Dr. Parisa Dehghan, Anahita Khosravi, and Fatemeh Dehghan Mongabadi for their sincere assistance.

Conflict of interest

“The author declare no conflict of interest.”