Advances in Application of PCR Amplification in Etiologic Diagnosis

Sixi YANG, Ran LI, Jinxia LIU

Pathogenic Biology Experimental Center, Chengde Medical College, Chengde 067000, China

Abstract In recent years, with the rapid development of molecular biology diagnostic technology, many new polymerase chain reaction (PCR) technologies with high specificity and good sensitivity have gradually been developed. While expanding the range of detection methods, these technologies inevitably have some disadvantages. Therefore, in clinical pathogen diagnosis, medical personnel should choose the detection method according to the detection purpose and pathogen characteristics. In this paper, the basic principle, application scope, advantages and disadvantages and development of various emerging PCR diagnostic techniques are respectively described in order to provide a theoretical reference for the selection of pathogenic biological diagnostic techniques in the clinical practice.

Key words New PCR, PCR amplification technology, Pathogen detection, Evaluation

1 Introduction

Etiologic diagnostic technology includes method methods such as traditional isolation culture and identification, molecular biology detection technology, and immunology detection technology. Polymerase chain reaction (PCR) amplification technology is a classic method for rapid amplification of pathogen nucleic acidinvitroaccording to the complementary base pairing rule. Due to high sensitivity and specificity, speed, efficiency, and good repeatability, the PCR technology has been widely applied in various fields such as medicine, biology, agronomy, and zoology. In this paper, we summarized and evaluated classic PCR amplification technologies and new PCR amplification technologies.

2 Classic PCR amplification technologies

The PCR technology invented by Mullisetal.[1]in 1986 is a milestone technology of rapidinvitroamplification of DNA or RNA fragments in nucleic acid diagnosis through the three stages of "denaturation-annealing-extension". The specific process is as follows: using DNA molecule as template, unwind the DNA double-strand into two single strands at 94 ℃, then gradually cool down to 55 ℃, make the primer specifically bind with the complementary sequence of the template DNA single strand, and then under the action of primers and DNA polymerase, they extend along the template strand according to the mechanism of semi-reserved replication until the synthesis of new DNA is completed. This technology has many advantages, such as high sensitivity and specificity, fast, high efficiency, good repeatability, easy operation, and wide applicability, but it also has many shortcomings. Pandaetal.[2]believed that the PCR amplification technology has some limitations in its application due to its extremely high sensitivity, which may lead to false positive results, and the experimental reagents and instruments used need high costs. In recent years, a variety of new PCR amplification technologies have been developed based on this technology to make them better used in the field of pathogenic diagnosis and epidemiological monitoring.

3 New PCR amplification technology based on the principles of molecular biology

3.1 Real-time fluorescence quantitative PCR (RFQ-PCR)

The real-time fluorescence quantitative PCR (RFQ-PCR) technology, vented in 1996 and based on the classic PCR amplification technology, combines PCR and fluorescence detection technology, and records the accumulation of fluorescent signal-labeled amplification products in each cycle of PCR amplification. In addition to the advantages of classic PCR amplification technology, the RFQ-PCR can also measure the initial concentration of target DNA in a wide dynamic range, has good reliability and reproducibility, high degree of automation, and accurate quantification, and it realizes a leap from qualitative to quantitative measurement. However, it is necessary to rely on the standard curve to calculate the target DNA copy number. Therefore, the diagnosis process is very complicated, requiring researchers to spend a lot of time to plot the standard curve, and it also needs supporting measuring instruments and result analysis devices. In 2018, Zhang Tieetal.[3]used the real-time fluorescence quantitative PCR amplification technology to detect infectious bronchitis virus nucleic acid, and the sensitivity was 10 times that of conventional PCR, RFQ-PCR can be used for epidemiological monitoring and pathogenic mechanism research of infectious bronchitis and it provides a new detection method for the prevention and control of infectious bronchitis.

3.2 Digital PCR (dPCR)Based on the research, Vogelstein[4]introduced the digital PCR (dPCR) in 1999. The dPCR technology divides the sample into a single molecule level, and then performs PCR amplification to obtain a digital (all or none) signal, to analyze the nature or number of the target molecule. It has the advantages of accuracy, high sensitivity and specificity, good repeatability, simplicity and practicality, low detection limit, wide application range and absolute quantification. It has following limitations: the PCR amplification product contaminates the environment and results in false positive results, the cost of reagents and instruments is high, and the operation process is very complicated. Due to these limitations, there are few clinical applications and fewer references. Blood microscopy is the gold standard for detectingBabesiamicrotiinfection. Nevertheless, this method is difficult to diagnose patients with parasitemia in the early or chronic stages of infection. Because the morphology ofB.microtiandPlasmodiumis similar, it requires professional and skilled technicians to make judgments. The droplet digital PCR (ddPCR) technology overcomes the above shortcomings to detectB.microtiandBabesiadunziin blood[5], and can be used as a highly specific and sensitive molecular detection method for human blood screening for babesiosis.

3.3 PCR-restriction fragment length polymorphism (PCR-RFLP)Based on the PCR, the PCR- restriction fragment length polymorphism (PCR-RFLP), amplifies a certain number of DNA fragments, which are digested by restriction enzymes to produce fragments of different sizes. Finally, they are separated by gel electrophoresis to obtain an RFLP pattern, so that the changes of single nucleotide can be detected. It is simple, cheap, fast, visual and comparable, and often used for typing and identifying pathogenic microorganisms. However, the sample size is small at one time and it needs highly specific primers. Human papillomavirus (HPV) is an important indicator of cervical cancer infection and prognosis evaluation. Treatment of cervical cancer based on HPV type is the key. There are many methods for clinical HPV typing, such as nucleic acid molecule rapid hybridization genotyping technology, cytology detection technology, pathology detection technology,etc.However, because there is still no gold standard for the classification of human papillomavirus (HPV), it is necessary to develop a new technology for clinical application and broaden the range of clinical alternative technologies. Golfettoetal.[6]developed PCR-RFLP to detect and type HPV in cervical cell samples. Because this method is cheap, fast and effective, it can be used as the main primary screening method for HPV in poor areas such as Brazil. Similarly, Héctoretal.[7]also applied this method for HPV typing in Argentina.

3.4 PCR-enzyme linked immunosorbent assay (PCR-ELISA)The PCR-enzyme linked immunosorbent assay (PCR-ELISA) is an immunoassay method that directly quantitatively detects PCR products after immobilizing biotinylated DNA on a microplate. Compared with the traditional PCR method, it has lower costs and simpler operation. This method combines PCR and ELISA as an analytical technique, and its application is very similar to ELISA, with qualitative and semi-quantitative capabilities, except that this method only allows the detection of proteins and not nucleic acids. Considering the above-mentioned advantages of PCR-ELISA, in recent years it has been applied to the species detection and identification of dermatophytes, the pathogen of tinea capitis in children, the identification and diagnosis of resistant Brucella bacteria[8], and the identification of the role of human papillomavirus (HPV) in oral cancer P16 positivity[9], the diagnosis of infants infected with and uninfected withLeishmania[10], and the selection and evaluation of PCR-ELISA for cutaneous leishmaniasis in Spain and Morocco[11].

3.5 Loop-mediated isothermal amplification (LAMP)The loop-mediated isothermal amplification (LAMP) is a new nucleic acid amplification technology that combines a set of 4 (or 6) different primers to 6 (or 8) different regions on the target gene. Under isothermal conditions, BST DNA polymerase amplifies a certain number of DNA copies into millions of copies in a short time. This technology derives many technologies, such as reverse transcription LAMP (RT-LAMP), multiple LAMP (mLAMP), electric LAMP (eLAMP), LAMP in optical disc drive (iD-LAMP),etc.This technology has the advantages of high specificity and sensitivity, simple operation, low cost, portability, and visualization of results, making LAMP suitable for rapid detection and identification of pathogens. However, its primer design is difficult, and it is easy to cause non-specific amplification to produce false positives. The result judgment is subjective and the generated DNA fragments are large. Therefore, this technology cannot be applied in cloning and other molecular biology fields. In 2015, Pooleetal.[12]developed a highly sensitive LAMP targeting the RF4 gene, which can detect 100 pg of DNA within 23-30 min, which is equivalent to 1 microfilaria. These studies have laid a foundation for LAMP in the field of filarial detection and the diagnosis of early infection.

3.6 Nanoparticle-assisted PCR (NanoPCR)In the PCR system, there are solid nanoparticles (1-100 nm in diameter) to form nanofluids. The thermal conductivity of nanofluids is greater than that of ordinary fluids, and the temperature can change extremely quickly, so as to reach the target temperature of the PCR technology more quickly. This technology has high sensitivity and specificity, is simple, efficient, and reproducible. However, after the specimen is subjected to nanoPCR, gel electrophoresis or sequencing of the amplified products is required for comparison, so it takes a long time. Yuan Wanzhe[13]first applied nanoPCR technology to detect porcine epidemic diarrhea virus (PEDV) in 2015, and compared this technology with conventional RT-PCR in terms of sensitivity and specificity: in terms of sensitivity, nanoPCR was 100 times more sensitive than conventional RT-PCR; in terms of specificity, nanoPCR did not amplify viral sequences other than PEDV. These prove that nanoPCR technology is a new method for detecting PEDV, and its detection results are reliable.

4 Immuno-PCR (i-PCR)

The immuno-PCR (i-PCR) is a new method of enzyme-linked immunosorbent assay (ELISA) combined with the sensitivity of nucleic acid amplification. This method is a qualitative and quantitative method for detecting the target antigen by PCR amplification of the DNA fragments marked with biotin avidin-conjugated antibodies, followed by electrophoresis analysis. The i-PCR is divided into many types of detection methods, mainly including: direct method, indirect method, double antibody sandwich i-PCR, double antigen sandwich i-PCR. For direct method: coat the antigen to be tested on a microtiter plate (microplate), bind the biotinylated antibody to it, and then combine the added streptavidinylated DNA with the biotinylated antibody, and finally detect whether it contains the target antigen by PCR amplification of the DNA; indirect method: detect antibody, coat the antibody on a microtiter plate, bine it to the biotinylated anti-antibody, and then combine the added streptavidinylated DNA with the biotinylated anti-antibody, finally detect whether it contains the target antibody by PCR amplification of the DNA; double antibody sandwich i-PCR: sandwich the antigen between the biotinylated capture antibody and the antibody to be tested, bind the capture antibody to the streptavidinylated DNA, and finally detect whether it contains the target antigen by PCR amplification of the DNA; double antigen sandwich i-PCR: sandwich the antibody between the biotinylated capture antibody and the antibody to be tested, bind the captured antigen to the streptavidinylated DNA, and finally detect whether it contains the target antigen by PCR amplification of the DNA. The advantages of i-PCR include high specificity and wide range of applications. It is recognized as one of the most sensitive detection methods for protein detection. It can also be used to detect cytokines, hormones, toxins and tumor markers. However, when the antigen concentration is low, it will not be detected, and when the sample contains a large amount of protein, it will cause false positive results. In addition, it requires washing and incubation, the automation is low, and it is easy to produce background signals.

Immuno-PCR is a new PCR amplification technology in recent years. Compared with other technologies, its application range is wider and its advantages are more obvious. Compared with immuno-PCR, real-time fluorescence quantitative PCR (RFQ-PCR) technology requires expensive fluorescent markers and measuring instruments, and has a narrower application range, and can only be used for nucleic acid detection; digital PCR is a relatively open system, the PCR amplification products obtained are prone to contamination, false positive results are obtained, and expensive detection equipment is required, there are few clinical applications due to the long detection time; PCR-RFLP is not a high-throughput method, and the samples tested each time are limited; PCR-ELISA is mainly used to detect proteins, such as antigens, antibodies,etc., and can also detect non-nucleic acid molecules, but cannot detect nucleic acids, and its application range is relatively narrow; LAMP needs to be designed as a set of primers, and problems with primer design can easily cause false positives in non-specific amplification, and the experimental process is prone to contamination, resulting in inaccurate results, so it can only be used for qualitative detection. The products obtained by nanoPCR need to be analyzed by sequencing or electrophoresis, which takes a long time. Similarly, this technology also has advantages over other pathogen detection technologies. Traditional separation culture and identification require smear microscopy and separation culture, which consumes manpower and material resources; single molecular biology detection technology requires expensive supporting facilities and relatively high cost; single immunological detection technology has low sensitivity and takes a long time.

In the late 1980s, Sanoetal.[14]combined PCR and enzyme-linked immunosorbent assay (ELISA) for the first time. At present, immuno-PCR technology has become more mature and can be used in such fields as agriculture, medicine, botany, zoology,etc.In the agricultural field, Ren Xianfengetal.[15]developed real-time immuno-PCR to detect total aflatoxin and zearalenone in grains. The method meets the requirements for simultaneous detection of multiple mycotoxins, and provides a broad application prospect for detecting mycotoxins in crops. In the field of zoology, Xie Quanetal.[16]established a new immuno-PCR method to detect avian leukemia virus (ALV) by applying the technique of adjacent ligation. This method is expected to become an effective method to detect ALV in clinic. In the medical field, in terms of fungi, Jiang Dongjianetal.[17]established a phage-based immunofluorescence quantitative PCR method for the detection of deoxynivalenol with high sensitivity and specificity; in terms of bacteria, Suman Sharmaetal.[18]established an indirect real-time immunopolymerase chain reaction (RTI-PCR) method based on immuno-PCR for the quantitative detection ofMycobacteriumtuberculosispst 1 (Rv 0934) in body fluids of tuberculosis patients. It is of far-reaching significance for the study of disease progression in different clinical stages of tuberculosis patients. In terms of bacteria, Suman Sharmaetal.[18]established an indirect real-time immunopolymerase chain reaction (RTI-PCR) method based on immuno-PCR for the quantitative detection ofMycobacteriumtuberculosispst 1 (Rv 0934) in body fluids of tuberculosis patients, which is of far-reaching significance for studying the disease progression of tuberculosis patients in different clinical stages.

5 Conclusions and prospects

With the continuous deepening of molecular biology technologies both at home and abroad, there have been considerable achievements in the PCR amplification technology. However, the currently new PCR amplification technology still has some problems to be solved, such as the improvement of technical sensitivity and specificity, the simplification of primer design and reaction conditions,etc.According to the above-mentioned development situations, PCR amplification technology should develop in the direction of high sensitivity, high specificity, low cost, high efficiency and simplicity. It is expected that in the near future, a more complete PCR amplification technology will be developed, so that it will be more and more widelyapplied, especially in basic medical research and clinical diagnosis.