**2.2 Loop-mediated isothermal amplification**

Nucleic acid amplification is commonly used in the field of life science research. With the development of molecular biology, many new molecular diagnostic technologies have been developed subsequently [11].

The loop-mediated isothermal amplification (LAMP-PCR) was first developed over 15 years ago, and it has emerged as powerful method to concurrently detect multiple pathogens [12, 13]. The method employs a DNA polymerase with strand displacement activity and a set of four inner and outer primers that recognize a total of six distinct sequences of the target DNA. Moreover, the method involves two successive steps of amplification, with the first step comprising mPCR and the second step LAMP. Amplicons of the first step serve as templates in the second step. The amplification protocol requires only a single temperature for the reaction, and the amplification is diagnosed without the need for electrophoretic techniques, using in situ detection process with colorimetric dye or with a fluorescent dye. The final products are the accumulation of 109 copies of target DNA in less than an hour. The LAMP-PCR has been regarded as an innovative technology and emerged as an alternative to PCR-based methodologies in clinical laboratory with significant increase of detection limits, efficiency, selectivity, and specificity over single-stage.

With more and more scientists focusing their attention on the application of LAMP technology, the range of its use is not limited to the bacteria detection and identification any more [14]. The LAMP-PCR was developed and employed to detect species that cause chorioamnionitis and premature labor, *Ureaplasma parvum*, and *Ureaplasma urealyticum* [15]*.*

It was also applied to the parasite and virus detection [16–18]. Recently, Kurosaki et al. in their study [19] in 2017 developed a LAMP-PCR assay for the detection of

Zika virus plasma, serum, and urine samples collected from 120 suspected cases of arbovirus infection in Brazil.

#### **2.3 Real-time PCR**

According to [20], clinical diagnostic approaches rely on quantitative PCR (qPCR) as a method to detect and quantify infectious agents. Fluorescence chemistry-based methods have revolutionized molecular diagnostics and become the gold standard for viral load quantification and detection of bacterial and viral pathogens. During qPCR, the nucleic acid is amplified until it produces a certain level of signal which is supplied through a DNA intercalating dye or sequence-specific fluorescent probe. The cycle threshold (Cq), defined as the number of amplification cycles required to reach that signal level, is used to calculate the number of target molecules originally present based on a standard curve [20]. In qPCR, the targets are detected in real time from the sealed PCR plate, and there is no post-PCR processing required; therefore, the risk of false-positive results due to amplicon carryover is substantially decreased compared to conventional [21].

During the past decade, advantages on PCR have become gold standard procedure in the diagnosis of infectious diseases, particularly the diagnosis of viral diseases, such as arboviruses. Arboviruses are causing an unprecedented health calamity in world, especially in Latin American countries, with rising statistics on a daily basis. The diagnosis of these diseases is difficult to establish only by clinical features. A substantial proportion of these infections are asymptomatic, but some patients may also present clinical symptoms similar to the arboviruses such as those caused by the Dengue, Zika, and Chikungunya virus. The qPCR technique is more sensitive and specific than serological tests. Besides that, in serum samples, it is only possible to perform the diagnosis in the acute phase of the disease that lasts up to 4–7 days after the onset of symptoms. In relation to the detection of Zika virus (ZIKV), for example, recent data from the literature indicate that in human urine, viral RNA was found longer compared to serum, up to 20 days after the onset of the first symptoms. In semen, studies show that the genetic material of ZIKV was found for weeks and even months after infection [22]. However, the determination of viral load presents some particular challenges when using qPCR methodology. This is because reliable absolute concentration results of qPCR are dependent on assay efficiency, instrument calibration metrics, and comparison with a known reference sample to convert the Cq measurements to a sample unknown.

#### **2.4 Digital PCR**

In contrast to qPCR, the digital PCR (dPCR) uses an alternative method that is not dependent upon the determination of the amplification cycle that the reporter dye signal exceeds a threshold. Instead, prior to amplification, the samples to be subjected to dPCR are divided into thousands of independent PCR reactions and are scored as either positive or negative for amplification of the viral sequence of interest. The positive wells are counted and converted to a target concentration in the original sample. This binary assignment of each reaction greatly minimizes the dependence of measurement on parameters such as the efficiency of the assay and the calibration of the instrument. Therefore, dPCR is the absolute quantification methodology with the greatest potential for quantification of low-load viral nucleic acids.

In the diagnostic routine, it is very common to obtain positive qPCR results obtained at the detection limit of this methodology, which may generate uncertainty of the result. dPCR is a complementary methodology that works beyond

**141**

isothermal conditions.

*PCR and Infectious Diseases*

diagnostic applications [23–25].

*Comparative analysis between RT-qPCR and dPCR.*

curve;

**Table 1.**

**3. Conclusions**

*DOI: http://dx.doi.org/10.5772/intechopen.85630*

the limit of detection of qPCR, since it is based on the Poisson distribution. Consequently, this methodology has a significant impact on research as well as on

**qPCR dPCR**

Quantification Relative quantification Absolut quantification, without standard

Factors affecting the signal Standard curve Results are not affected by any parameter

curve

• Reduction of the impact of inhibitors present on complex samples.

• Quantification of viral genomes in samples without the need to use a standard

**Table 1** shows a comparison between the two methodologies: qPCR and dPCR. A great number of studies use dPCR to diagnose infectious disease-related viruses, including hepatitis B virus (HBV), cytomegalovirus, human influenza, and HIV [26–28]; bacterial infections (*Mycobacterium tuberculosis*, *Chlamydia trachomatis*, and *Staphylococcus aureus*) [29–31]; and parasitic infectious such as detection of

The ability to concurrently detect multiple pathogens infecting a host is crucial for accurate diagnosis of infectious diseases, identification of coinfections, and assessment of disease state for an effective patient management. PCR technology has been widely used to detect and quantify pathogenic microorganisms that cause various infectious diseases including some arboviruses, STIs, and bacterial infection. This methodology is revolutionizing the area of molecular diagnostics because of its high sensitivity of detection and specificity for the determination of infectious agents. In addition, there is a reduction in run time and cost over traditional cultivation methods, for example, to determine the amount of a particular pathogen in a clinical specimen. The main advantages of PCR are its higher sensitivity and specificity compared with other diagnostic methods such as serological assays and culture methods, as well as its rapidity, utility, and versatility in clinical laboratory. Although the conventional PCR is the most widely used molecular technique, other methodologies have been developed including real-time PCR, multiplex PCR, LAMP-PCR, and digital PCR. The biochemical mechanisms of these techniques are based on enzyme-mediated processes, target, signal or probe amplification, and

Some benefits of digital PCR in virology can be cited [25]:

Results Cq, ∆Cq, or ∆∆Cq Copies/mL

Instrument Primers and probes

• Detection of viruses that have a very low viral load;

• Use of low concentration of samples;

*Plasmodium falciparum* and *Plasmodium vivax* [32].


#### **Table 1.**

*Synthetic Biology - New Interdisciplinary Science*

arbovirus infection in Brazil.

**2.3 Real-time PCR**

Zika virus plasma, serum, and urine samples collected from 120 suspected cases of

According to [20], clinical diagnostic approaches rely on quantitative PCR (qPCR) as a method to detect and quantify infectious agents. Fluorescence chemistry-based methods have revolutionized molecular diagnostics and become the gold standard for viral load quantification and detection of bacterial and viral pathogens. During qPCR, the nucleic acid is amplified until it produces a certain level of signal which is supplied through a DNA intercalating dye or sequence-specific fluorescent probe. The cycle threshold (Cq), defined as the number of amplification cycles required to reach that signal level, is used to calculate the number of target molecules originally present based on a standard curve [20]. In qPCR, the targets are detected in real time from the sealed PCR plate, and there is no post-PCR processing required; therefore, the risk of false-positive results due to amplicon

During the past decade, advantages on PCR have become gold standard procedure in the diagnosis of infectious diseases, particularly the diagnosis of viral diseases, such as arboviruses. Arboviruses are causing an unprecedented health calamity in world, especially in Latin American countries, with rising statistics on a daily basis. The diagnosis of these diseases is difficult to establish only by clinical features. A substantial proportion of these infections are asymptomatic, but some patients may also present clinical symptoms similar to the arboviruses such as those caused by the Dengue, Zika, and Chikungunya virus. The qPCR technique is more sensitive and specific than serological tests. Besides that, in serum samples, it is only possible to perform the diagnosis in the acute phase of the disease that lasts up to 4–7 days after the onset of symptoms. In relation to the detection of Zika virus (ZIKV), for example, recent data from the literature indicate that in human urine, viral RNA was found longer compared to serum, up to 20 days after the onset of the first symptoms. In semen, studies show that the genetic material of ZIKV was found for weeks and even months after infection [22]. However, the determination of viral load presents some particular challenges when using qPCR methodology. This is because reliable absolute concentration results of qPCR are dependent on assay efficiency, instrument calibration metrics, and comparison with a known reference

In contrast to qPCR, the digital PCR (dPCR) uses an alternative method that is not dependent upon the determination of the amplification cycle that the reporter dye signal exceeds a threshold. Instead, prior to amplification, the samples to be subjected to dPCR are divided into thousands of independent PCR reactions and are scored as either positive or negative for amplification of the viral sequence of interest. The positive wells are counted and converted to a target concentration in the original sample. This binary assignment of each reaction greatly minimizes the dependence of measurement on parameters such as the efficiency of the assay and the calibration of the instrument. Therefore, dPCR is the absolute quantification methodology with the greatest potential for quantification of low-load viral nucleic

In the diagnostic routine, it is very common to obtain positive qPCR results obtained at the detection limit of this methodology, which may generate uncertainty of the result. dPCR is a complementary methodology that works beyond

carryover is substantially decreased compared to conventional [21].

sample to convert the Cq measurements to a sample unknown.

**140**

acids.

**2.4 Digital PCR**

*Comparative analysis between RT-qPCR and dPCR.*

the limit of detection of qPCR, since it is based on the Poisson distribution. Consequently, this methodology has a significant impact on research as well as on diagnostic applications [23–25].

Some benefits of digital PCR in virology can be cited [25]:


**Table 1** shows a comparison between the two methodologies: qPCR and dPCR. A great number of studies use dPCR to diagnose infectious disease-related viruses, including hepatitis B virus (HBV), cytomegalovirus, human influenza, and HIV [26–28]; bacterial infections (*Mycobacterium tuberculosis*, *Chlamydia trachomatis*, and *Staphylococcus aureus*) [29–31]; and parasitic infectious such as detection of *Plasmodium falciparum* and *Plasmodium vivax* [32].
