Menstrual blood (MB) is a potential sample for gynecological diseases diagnosis: MB flows through female reproductive tract, carrying many different biomarkers related to infection or other diseases; MB is easy to collect (using pad or tampon); and the cost for sampling is nearly zero. These advantages make MB “main character” in our project (Figure 1).

Figure 1. Detect HPV using menstrual blood. An overview of our project.

Researches, though limited in number, showed that the agreement rate between traditional HPV testing and MB HPV testing ranged from 87.5%^[1] to 97.8%^[2]. The sensitivity of MB HPV testing is 80%^[2-4] or so, and the specificity is higher than 90%^[2-4]. Therefore, using MB for HPV screening is totally possible and reliable theoretically as well as experimentally.

Only 15 subtypes of HPV are high-risk strains which are able to cause cancer, they are HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82. Considering that the typing of HPV were based on their L1 sequences in its genome and it encodes a capsid proteins which has no virulence or toxicity, we decided to use it as our target.

Plan A. CRISPR/Cas12a Biosensing System

In order to make the usage of PaDetector as convenient as possible, we refer to CRISPR/Cas biosensing system, which is expected to become an expedite way of disease diagnosis.^[5] In the process of designing our own detection method, we use SHERLOCKv2 and DECTRT as reference.^[6,7] Here are an overview of this method:

After getting the raw sample, we firstly inhibit RNase activity and lyse cells with ddh3O.Then, the signal (HPV genome) will be amplified with recombinase polymerase amplification (RPA) technology (for RNA, RT-RPA). After that, Cas12a combined with specified crRNA will recognize the amplified signal and exhibits a “collateral effects” of promiscuous DNase activity and cut the single-strand DNA reporter. Finally, get the result by checking if the reporter is cut (Figure 2).

Figure 2. Process of target detection with our Cas12a biosensing system.

Primary signal amplification with RPA

RPA use recombinase to guide primers to the target sequence and pair their bases. After that, DNA polymerase will start replication, single strand will be bound by SSB to make sure the smooth running of this reaction.

Figure 3. Process of recombinase polymerase amplification (RPA).

The main differences between RPA and PCR are: 1) RPA can be carried out at low temperature. Generally, the RPA reaction is carried out at 37℃. In our project, however, the temperature was lower to 25℃ in order to ensure that our product do not require any kind of heating device. 2) In the process of RPA reaction, there is no “cycle”, the amplification reaction is continuous. We deeply analyse this phenomenon in the modelling part. Click here to learn more.

Secondary signal amplification with Cas12a

After 40-60 min of RPA reaction, the target is adequately amplified, and Cas12a as well as a specific crRNA will be introduced into the reaction mix. Cas12a firstly combined with crRNA and if the target was present, crRNA will pair with it so that Cas12a can “recognize” the target (Figure 4) and exhibits a “collateral effects” of promiscuous DNase activity.

Figure 4.3D structure of FnCas12a (grey), shows the crRNA (beige) which binds the complementary target double strand DNA (brown).
Picture from SWISS-MODEL.

Detect Cas12a’s DNase activity

The last step is to detect the twice amplified signals. In our project, we provide different way of reporting by changing the reporter in order to cater different needs.

The first kind of reporter we use is a 5bp single strand DNA with 5’-Biotin and 3’-Fitc. With this kind of reporter, the lateral flow detection is carried out to show whether the reporter was cut which reflects the Cas12a’s DNase activity.

Figure 5. Schematic of lateral-flow detection test paper (right) and two possible test results (left): Negative result, only Control line is visible, means that Cas12a remains inactive and the reporter is uncut; Positive result, both Control line and Test line appear, suggest that the reporter is cut.

The schematic of the lateral-flow detection of PaDetector is shown in Figure 5. Here, the control line is composed of Fitc antibody, whose molar amount is more than one time of reporter. Besides, molar amount of the reporter is more than one time of Au-Strep but less than four times of it.

After the reaction, drop the reaction liquid onto the sample pad of the test paper. Then, the liquid flows towards the absorption pad (shown in Figure 4). Firstly, the reporters’ 3’-Biotin combine with the Au-Streps in the test paper. Because reporters are excessive, all the Au-Streps can be combined with the reporter. After that, the reporter combined with Au-Streps flow through the Control line, where the Fitc antibodies will link to 5’-Fitc specifically, so if Cas12a is not activated and all the reporters remain uncut, the reporter will stop at the Control line with the Au-Streps combined with them, while the Test line would be invisible because no Au-Strep can reach there, indicate that the sample is free from a specific subtype of HPV.

Otherwise, the result is positive when which Cas12a is activated and reporters are cut, Au-Streps will not stop at the Control line (at least most of them will not). Correspondingly, the colour of the Control line will be invisible or at least lighter and Au-Streps will continue to flow forward and combine with the Biotin at Test line, thus accumulated to show a band.

The principle advantage of the lateral-flow detection is that the test result can be checked by naked eyes.

The second kind of reporter we use is a 5bp single strand DNA with 5’-FAM and 3’- TAMRA (Figure 6).^[8] FAM is a kind of fluorescent group, and its fluorescence can be quenched by TAMRA. Thus, when Cas12a is not activated and reporter remain uncut, the fluorescence of the reporter’s 5’-FAM is quenched by its 3’-TAMRA, therefore, there will be no fluorescence in the reaction system. When Cas12a is activated and the reporters are cut, FAM’s fluorescence will recover, indicate that the sample contains a specific subtype of HPV.

Figure 6. Schematic of fluorescent reporter detection.

The advantage of using fluorescent group is that we can monitor the reaction process by fluorescence reading instrument, thus, we can determine the time required and compare the effectiveness of different Cas12a and crRNA.

Expanding the application scope

Cas12a biosensing system is highly portable, by simply changing the target sequence of crRNA, we will be able to detect different kinds of diseases, from virus infection to genetic disorders, or even cancers. Considering this, we hope to apply it to different kinds of diseases detection. Take our PaDetector for example, by simply changing the RPA primers and crRNA target sequence, PaDetector would be able to detect different kinds of gynecological infection or other gynecological diseases. Thus, the only problem is to find biomarkers for the disease users are interested in. In the present world, many different databases for all sorts of biomarkers have already been established with clear organization, comprehensive data and powerful search function. However, different users always want different kinds of biomarkers for diverse purpose, there will never be a biomarker database that can satisfy every user. That’s why we develop a Database Management System (DBMS) for every researcher who wants to establish a localized database which can meet their specific needs exactly, such as a biomarker database for gynecological diseases for PaDetector. With this biomarker DBMS, you will be able to manage your biomarkers just like using EndNote to manage your references. It would be fantastically convenient and extraordinarily customized.

We have uploaded this DBMS to GitHub, we just provide a demo and we do hope that it will be updated and perfected by future iGEM teams. Learn more in Software.

Plan B. Hybridization Chain Reaction Biosensing System

After putting forward the detection method based on CRISPR-Cas12a, we went to the hospital and had a conversation with Prof. Luv. He suggested that we could design a project to do multi-channel detection of HPV. Since HPV16 and HPV18 are the most high-risk subtypes, we choose these two subtypes as our target, and aim at distinguish them in one reaction system without heating or any other laboratory operations, which indicates that our method has the potential to detect and distinguish multiple subtypes of HPV simultaneously. Taking these into consideration, we come up with the detection method based on hybridization chain reaction. This method includes 3 reactions, exonuclease III (Exo III)-assisted signal amplification, hybridization chain reaction (HCR) and lateral flow nucleic acid biosensor (LFNAB), which all have been reported to show great potential in the field of amplification detection^[9-11].

Version 1.0

In version 1.0, we plan to enhance the HPV signal through a linear Exo III-assisted signal amplification.

Figure 7. Hybridization Chain Reaction Biosensing System – Version 1.0

Preprocessing of HPV genome (Figure 7A)

Firstly the HPV genome is digested with two kinds of endonucleases, which will produce a double strand DNA (dsDNA) with only one recessed 3’-end. Then Exo III digests the specific fragment with its exonuclease activity to the recessed 3’-terminus in dsDNA, so the target single strain DNA (ssDNA) which we call signal 1 (S1) is released.

First stage liner signal amplification: Exo III-assisted signal amplification (Figure 7B)

S1 binds to the stem loop probe 2 (P2) through complementary pairing of partial bases, and P2-S1 will be digest by Exo III and produce signal 2 (S2). This causes the first stage linear signal amplification.

P2 is a ssDNA that forms a stem loop through denaturation and annealing, which is previously prepared in the reaction system. It has a 5’- FITC or Cy3 modification for further detection on the test paper, and has a modification of thiophosphorylation at a specific site to avoid ExoIII’s digestion. It is generally assumed that ExoIII can only cut from recessed or blunt 3’-end of dsDNA, but in our experiment, ExoIII also has the potential to degrade single-stranded DNA, and it also has been mentioned in some literatures^[12]. Meanwhile, many literatures also mentioned that Exo III cannot digest the thiophosphorylation modified site^[13], so we make some of our probe thiophosphorylation modified in order to prevent the nonspecific digestion by Exo III.

Second stage signal amplification: Hybridization chain reaction (Figure 7C)

S2 can cause a hybridization chain reaction with H1 and h3, and produce a hybridized chain contains one FITC or Cy3 and several biotins. H1 and h3 both have a 3’-thiophosphorylation modification to avoid ExoIII’s digestion, and a 5’-biotin modification for further detection on test paper. For different subtypes of HPV, we can get different S1, and for different S1 we can design to produce S2 with different modification. For example, for detection of HPV16, we will design to produce S2 with FITC modification, and for HPV18, we will designed to produce S2 with Cy3 modification.

Test paper: Lateral flow nucleic acid biosensor (Figure 7D)

When the reaction system is put into LFNAB, one hybridization chain can bind several AuNPS-Strep. S2 is 5’- modified with specific fluorophore so it can bind to the corresponding antibody on the ‘test line’ of our test paper. The test paper will show two lines (one ‘test line’ and one ‘control line’) in the presence of one kind of S2, and it will show three lines (two ‘test line’ and one ‘control line’) in the presence of two kinds of S2. We can develop different systems in which different endonucleases can produce different S1, and different S1 can produce different S2 which is 5’- modified with other desirable proteins so that we can realize multiple screening for HPV.

Version 2.0

In version 2.0, we try to replace the signal amplification process with index increase in order to make our detection more sensitive. We refer to an ExoIII-assisted cascade signal amplification strategy for label-free and ultrasensitive chemiluminescence detection method raised by Gao Y et.al in 2014^[14].

Figure 8. Hybridization Chain Reaction Biosensing System – Version 2.0

In this design, S1 is generated in the same way as version 1.0 (Figure 8A). Then S1 hybridizes with probe 1-signal 2(P1-S2) which is previously prepared through denaturation and annealing, then the hybrid probe (S1-P1-S2) is digested by Exo III and S1, S2 and signal 3 (S3) are released. S3 can also hybridize with other P1-S2 and release S2 because of its same sequence with S1. The increasing number of S3 is able to produce more and more S2, which causes the first stage exponential signal amplification (Figure 8B). And our S2, H1 and h3 are also 3’- phosphorothioate modified in order to prevent the nonspecific digestion of ssDNA by Exo III.

The second stage signal amplification: Hybridization chain reaction (Figure 8C) and lateral flow nucleic acid biosensor (Figure 8D) in version 2.0 is the same with version 1.0. Finally, the reaction system will pass through the test paper containing two ‘test lines’ and one ‘control line’. The coloration of both the ‘control line’ and any ‘test line’ shows a positive result, and only the coloration of the ‘control line’ shows a negative result. In this way, multiple detection of HPV16 and HPV18 can be realized on a strip of test paper.

Expanding the application scope of hybridization chain reaction biosensing system

Hybridization chain reaction biosensing system is highly alterable, by simply changing the restriction endonuclease and the sequence of P2 or P1, we will be able to detect different DNA targets, from virus infection to genetic disorders, or even cancers. Considering this, we hope to apply it to different kinds of diseases detection. Take our PaDetector for example, by simply changing the restriction endonuclease and the sequence of P2 or P1, PaDetector would be able to detect different kinds of gynecological infection or other gynecological diseases. Thus, the only problem is to find biomarkers for the disease users are interested in. In the present world, many different databases for all sorts of biomarkers have already been established with clear organization, comprehensive data and powerful search function. However, different users always want different kinds of biomarkers for diverse purpose, there will never be a biomarker database that can satisfy every user. That’s why we develop a Database Management System (DBMS) for every researcher who wants to establish a localized database which can meet their specific needs exactly, such as a biomarker database for gynecological diseases for PaDetector. With this biomarker DBMS, you will be able to manage your biomarkers just like using EndNote to manage your references. It would be fantastically convenient and extraordinarily customized.

We have uploaded this DBMS to GitHub, we just provide a demo and we do hope that it will be updated and perfected by future iGEM teams. Learn more in Software.

Reference

[1] Lee, B. et al. Detection of high-risk human papillomavirus using menstrual blood in women with high-grade squamous intraepithelial lesions or high-risk human papillomavirus infections: A pilot study. Journal of Obstetrics and Gynaecology Research 42, 319-324.

[2] Kim, S. R. et al. Pad – a new self-collection device for human papillomavirus. International Journal of STD & AIDS 18, 163-166.

[3] Wong, S. C. C. et al. Human papillomavirus DNA detection in menstrual blood from patients with cervical intraepithelial neoplasia and condyloma acuminatum. J Clin Microbiol 48, 709-713.

[4] Budukh, A. et al. Menstrual pad, a cervical cancer screening tool, a population-based study in rural India. Eur J Cancer Prev 27, 546-552.

[5] Li, Y., Li, S., Wang, J. & Liu, G. CRISPR/Cas Systems towards Next-Generation Biosensing. Trends in Biotechnology 37, 730-743.

[6] Chen, J. S. et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436-439.

[7] Gootenberg, J. S. et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360, 439-444.

[8] Gootenberg, J. S. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438-442.

[9] Liu, W. J., Xu, Q., Ma, F., Li, C. C. & Zhang, C. Y., Exonuclease III-assisted multiple cycle amplification for the sensitive detection of DNA with zero background signal. ANALYST 143 5461 (2018).

[10] Lu, S., Hu, T., Wang, S., Sun, J. & Yang, X., Ultra-Sensitive Colorimetric Assay System Based on the Hybridization Chain Reaction-Triggered Enzyme Cascade Amplification. ACS Appl Mater Interfaces 9 167 (2017).

[11] Yin, R. et al., A validated strip-based lateral flow assay for the confirmation of sheep-specific PCR products for the authentication of meat. FOOD CONTROL 60 146 (2016).

[12] Yang, Z., Sismour, A. M. & Benner, S. A., Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages. NUCLEIC ACIDS RES 35 3118 (2007).

[13] Xu, Q., Cao, A., Zhang, L. F. & Zhang, C. Y., Rapid and label-free monitoring of exonuclease III-assisted target recycling amplification. ANAL CHEM 84 10845 (2012).

[14] Gao, Y. & Li, B., Exonuclease III-Assisted Cascade Signal Amplification Strategy for Label-Free and Ultrasensitive Chemiluminescence Detection of DNA. ANAL CHEM 86 8881 (2014).

Team:ZJU-China/Design

Detect HPV using menstrual blood