Development and Clinical Evaluation of a CRISPR-Based Diagnostic for Rapid Group B Streptococcus Screening

Lingxiao Jiang; Weiqi Zeng; Wanting Wu; Yingying Deng; Fusheng He; Wenli Liang; Mingyao Huang; Hong Huang; Yongjun Li; Xiaorui Wang; Hang Su; Shilei Pan; Teng Xu

Disclosures

Emerging Infectious Diseases. 2021;27(9):2379-2388. 

In This Article

Materials and Methods

Study Participants and Sample Collection

A total of 426 pregnant women were prospectively admitted into Zhujiang Hospital (Guangzhou, China) for antenatal care during March 7–November 22, 2019. We excluded 14 from this cohort study because of insufficient samples for testing, incomplete clinical or experimental data, or invalid test results attributable to internal control failures. We included the remaining 412 samples in the prospective cohort study, in which direct culture, direct clinically validated PCR, and CRISPR-GBS tests were performed for each patient.

We conducted the validation cohort retrospectively, where we performed direct culture and CRISPR-GBS. For the purpose of validation, we included for enrichment culture 31 samples consisting of about one third each of dual-positive, dual-negative and discordant samples, according to the results of direct culture and CRISPR-GBS.

We collected vaginal–rectal swab specimens from the enrolled patients. Sample collection was reviewed and approved by the Zhujiang Hospital Ethics Committee Review Board. Informed consents were signed by patients or their surrogates.

Cas13a Protein

After codon optimization, we synthesized the open reading frame (ORF) of Cas13a and cloned it by using Gene Services (Genscript Biotech, https://www.genscript.com). The Cas13a ORF expression vector was transfected into Escherichia coli BL21. We first grew transfected cells at 37°C and then incubated them with isopropyl β-d-1-thiogalactopyranoside at 16°C. We purified proteins from lysed bacteria by using the Ni-NTA protocol[18] and stored aliquots of purified protein at −80°C.

Strains and Human DNA

We purchased the S. agalactiae (group B Streptococcus) strain from the American Type Culture Collection (ATCC13813). S. pneumoniae, S. pyogenes, S. mitis, Enterococcus faecalis, Acinetobacter baumannii, and Pseudomonas aeruginosa strains were donated by China's National Institutes for Food and Drug Control. We purchase another 2 species of bacteria, E. coli and Staphylococcus aureus, from China's General Microbiological Culture Collection Center. We purchased pure human DNA from Solarbio (http://www.solarbio.net), which we eluted in nuclease-free water.

Oligos and gRNA

Primer with an appended T7 promoter used in the recombinase polymerase amplification (RPA) for atoB amplification were forward primer 5′-TAAT ACGA CTCA CTAT AGGG AATT GAAT GGAA TGAA CCAT TTGC AGCG AT-3′ and reverse primer 5′-AATA ATTC CTGA GCAG GCAT AAGG GTGT C-3′. We used sgRNA for Cas13 (5′-GGGG AUUU AGAC UACC CCAA AAAC GAAG GGGA CUAA AACU CUCU CUUC AGGA UAAU AAUG AUUA AAU-3′) and ssRNA probe (5′-6-FAM-UUUUUC-BHQ1) for CRISPR detection after RPA amplification. Primer used in the nested PCR amplification for atoB amplification for round 1 were forward primer 5′-ACGG AAAA ACTA TTAA CAGA AACT CATA CT-3′ and reverse primer 5′-AATA ATTC CTGA GCAG GCAT AAGG GTGT C-3′ and for round 2 were forward primer 5′-CTCA TACT AAAA TATC GGAT TATG ATGC-3′ and reverse primer 5′-AGGC ATAA GGGT GTCC GTAA GC-3′.

DNA Rapid Extraction

We eluted swabs with 1 mL of saline. We transferred 200 μL of eluate to a new sterile, nuclease-free 1.5-mL tube. After a 5-minute centrifugation at 10,000 × g, we resuspended the pellet in lysis buffer consisting of 0.1% sodium dodecyl sulfate and 1% NP40. We added glass microbeads and used a Crystal Industries vortex mixer (https://crystalindustries.com) to disrupt the bacterial cell walls. We then heated samples at 99°C for 10 min and centrifuged them again at 14,000 × g. We used 2 μL of supernatant as template for each subsequent assay for GBS detection.

CRISPR-GBS

The CRISPR-GBS test combines an RPA step and a subsequent T7 transcription and Cas13 detection step, as described previously.[17] In brief, we incubated reactions containing 2 μL of sample, 0.4 μM of each primer, 1 × reaction buffer, 14 mM of magnesium acetate, and the RPA enzyme mix at 37°C for 30 min. Then we added the amplification product to the CRISPR reaction mix, consisting of 33.3 nM of gRNA, 66.7 nM of Cas13, 5 mmol/L of each nucleotide triphosphate, 1 μL of T7 RNA polymerase (New England Biolabs, https://www.neb.com) and 166 nM of ssRNA reporter. We incubated the final reaction mix at 37°C and monitored it for fluorescence signal. We collected fluorescent signals by using an ABI7500 qPCR machine (ThermoFisher Scientific, https://www.thermofisher.com) for 20 min.

Evaluation of Limit of Detection

For the evaluation of limit of detection by the number of genomic copies, we purified DNA of the GBS strain (ATCC13813) and determined the concentration by using Qubit (ThermoFisher Scientific). We calculated the number of genomic copies by using the formula

We performed serial dilution with nuclease-free water to achieve desired concentrations. For the evaluation of limit of detection by CFU per mL, we serially diluted a reference ATCC strain with known CFU with a negative sample to the desired titer before subjecting it to DNA extraction. Although accurate conversion is challenging, our and others' observations comparing DNA quantity and CFU counts showed that 1 CFU equaled ≈3–5 genome copies (data not shown).[19]

We used 2 μL of extracted DNA at each titer as templates. We performed 10 replicates at each data point.

Direct Culture and Enrichment Culture

We eluted each swab with 1 mL of saline. For direct culture, we inoculated 200 μL of eluate onto selective chromogenic GBS screening media (CHROMID Strepto B; bioMérieux, https://www.biomerieux-diagnostics.com) and incubated it at 37°C for 24 h aerobically. We incubated negative plates for another 24 h before the final plate reading. For enrichment culture, we first inoculated 200 μL of swab eluate into selective Todd Hewitt broth and incubated it at 37°C aerobically overnight. We then inoculated the enriched broth onto chromogenic Brilliance GBS agar (bioMérieux) by using the same experimental procedures as direct culture. We subjected all suspect colonies to Lancefield streptococcal grouping to confirm GBS.

PCR and Nested PCR

We performed the regular PCR testing by using a validated commercial GBS PCR kit (BEC, http://www.biochainbj.com) according to the manufacturer's instructions. We performed the nested PCR assay in 2 successive rounds of amplification. The first round amplified a larger fragment of the atoB gene for 35 cycles. We then subjected 2 μL of the primary PCR product to the second amplification by using a nested set of primers targeting a shorter fragment as part of the first amplicon. We then purified the amplicons from the second round and subjected them to Sanger sequencing for validation. We considered positive only those samples that both yielded PCR products after the second round of amplification and had sequences validated by Sanger.

Statistical Analysis

We conducted comparative analysis by using Pearson χ 2 test, Fisher exact test, or the Student t-test, where appropriate. We performed data analyses by using SPSS Statistics 22.0 (IBM, https://www.ibm.com). We considered p values <0.05 as statistically significant. All tests were 2-tailed unless indicated otherwise.

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