Human Papillomavirus Detection Method

Detection by Morphological Methods

Since HPV cannot proliferate in vitro, the morphological method originated in the 1940s is a traditional method, which mainly includes Pap smear cytopathological detection, electron microscopy technology (direct observation of viral particles), etc.

Figure 1. Diagnostic methods for detection of HPV infection.(Bogusiak K, et al., 2014)

• Pap Smear Cytopathology Test

Hollow cells are the main morphological changes of HPV infection. The diagnosis can be made if the hollow cells caused by HPV can be seen by Pap staining in the cervical transition zone. There are many factors that affect Pap smears, and the false negative rate is high, which limits its application. In recent years, with the advancement of specimen collection, production, and reading technology, traditional Pap smears have developed into liquid-based thin-layer cell technology (TCT) and automatic smear detection systems. TCT has relatively small impact, The operation is standardized, and its detection accuracy is higher than that of conventional Pap smear, and it is used clinically for large-scale census and screening. Since other viral infections and human factors may cause vacuoles or hollow changes in cells, as well as the influence of factors such as material sampling, staining, and subjective judgment, morphological detection of HPV still has low sensitivity, poor specificity, and false positives. It has the disadvantages of high negative rate and false positive rate, and it cannot classify HPV.

• Electron Microscopy Technology

Types of colposcopes with electron microscopy technology include: traditional optical colposcope, photoelectric integrated colposcope and electronic colposcope. HPV is one of the earliest viruses to be observed under an electron microscope. The biopsy specimen is negatively stained. If characteristic virus particles can be seen under an electron microscope, a diagnosis can be made. Although electron microscopy is highly accurate, it is time-consuming, expensive instruments and equipment, and has a low positive rate. The specificity and sensitivity of electron microscopy for detecting HPV are not high, and it is not convenient to classify HPV, so it is currently rarely used.

Molecular Biology Methods

• PCR

Polymerase chain reaction (PCR) detects HPV-DNA. The most critical factor in evaluating HPV detection methods is detection sensitivity. Detection of HPV DNA based on gene amplification is currently the most sensitive detection method. PCR can not only be used to detect viral load quantification, DNA sequencing and mutation analysis, but can also perform multiplex amplification and analyze multiple DNA sequences simultaneously, and PCR has the characteristics of relatively simple operation, time saving, and unrestricted specimen sources. Therefore, PCR Testing is currently the best method for HPV-DNA testing and typing. Its disadvantage lies in its high sensitivity, which can easily lead to false positive detection due to cross-contamination of samples. The operation of PCR typing is relatively cumbersome, and the number of samples tested at one time is limited, which limits its application in large-scale screening.

• Nucleic Acid Molecular Hybridization Technology

Dot hybridization or in situ hybridization is used to detect HPV-DNA based on different specimens, which has good specificity and sensitivity. Nucleic acid hybridization detection methods mainly include in situ hybridization (ISH), nucleic acid blotting, hybridization capture (HC), etc. ISH uses HPV probes to hybridize to HPV-DNA in tissues. Its advantage is beneficial to pathological analysis, but the stability of the hybrid chain is not high. During the hybridization process, some probes may be single-stranded, reducing the hybridization rate and affecting the detection rate of HPV-DNA. Nucleic acid blotting is suitable for HPV typing and HPV-DNA relative molecular mass identification. It was used in early research on HPV, but it requires fresh tissue specimens and is complicated to operate. HC is a new method in the 21st century. Its basic principle is to use efficient liquid-phase RNA-DNA hybridization method to capture HPV-DNA in the sample. An alkaline phosphatase-labeled anti-RNA-DNA antibody-chemiluminescent signal display system was used.

HC-I can detect 9 high-risk HPV types, but its detection sensitivity is lower than PCR and other gene amplification technologies, and its clinical application is limited. Currently, HC-II is widely used to detect viral DNA and has a high negative predictive value for cervical lesions and cervical cancer. This method has been recognized worldwide and is widely used for screening and review of cervical cancer.

• Gene Chip Technology

Gene chip technology is a new technology that was developed at the end of the 20th century. Its principle is to use in-situ synthesis or direct spotting methods to arrange a large number of DNA fragments or oligosaccharide nucleotide fragments on the surface of a support to produce two DNA fragments. The DNA probe array is then hybridized with the labeled sample, and the quantity and sequence information of the sample molecules are obtained by detecting the intensity of the hybridization signal, so as to study the gene sequence and function. The gene chip method has the advantages of miniaturization, intensification and standardization, and high throughput. The sensitivity and specificity of gene chips are higher than other methods. It can not only be used for typing, but also detect multiple types of mixed infections at the same time. However, due to the high cost of detection, it is difficult to put it into practical clinical application. However, with the development of this technology, if the cost decreases, it will undoubtedly be the most potential detection and analysis method in the future.